Archive for the ‘Crispr’ Category

1987: CRISPR repeats were observed in bacterial genomes. The authors concluded, no sequence homologous to these has been found elsewhere in procaryotes, and the biological significance of these sequences is not known. Ishino et al. J. Bacteriology (1987) 169:5429-5433. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC213968/

2002: The term CRISPR was coined to describe the repetitive repeats observed in bacterial and archaeal genomes. Genes usually found associated with the CRISPR repeats were identified and named CRISPR Associated Proteins or Cas. Jansen et al. Mol. Microbiology. (2002) 43:1565-1575. http://www.ncbi.nlm.nih.gov/pubmed/11952905

2005: CRISPR spacer sequences were matched to foreign DNA. Bolotin et al. Microbiology (2005) 151:2551-2561. http://www.ncbi.nlm.nih.gov/pubmed/16079334

2006: CRISPR was first proposed to be a bacterial adaptive immune system. Makarova et al. Biol Direct (2006) 1:7. http://www.ncbi.nlm.nih.gov/pubmed/16545108.

2007: CRISPR loci were found to impart phage resistance in bacteria. It was determined that CRISPR sequences together with the Cas genes impart resistance and that resistance to specific phages was determined by the spacer sequences found between CRISPR repeats. Barrangou et al. Science. (2007) 315:1709-1712. http://www.ncbi.nlm.nih.gov/pubmed/17379808

2009: RNA guided RNA cleavage is first described. Hale et al. RNA (2008) 2:2572-2579. http://www.ncbi.nlm.nih.gov/pubmed/18971321

2010: The CRISPR/Cas system was identified as a bacterial and archeal immune system that targets and cleaves phage DNA. This system was found to be dependent on the bacteria containing CRISPR spacer sequences that match the phage DNA. Additionally researchers discovered that new spacer sequences could be inserted into the bacterial/archeal chromosome making the CRISPR/Cas system an adaptive immune system. Garneau et al. Nature. (2010) 468:67-71. http://www.ncbi.nlm.nih.gov/pubmed/21048762

2011: Cas9 from Streptococcus pyogenes was found to associate with two RNA molecules coined crRNA and tracrRNA and that all these components are required for protection against phage infection. Deltcheva et al. Nature (2011) 471:602-607. http://www.ncbi.nlm.nih.gov/pubmed/21455174

2012: Cas9 was found to be an endonuclease capable of introducing DSB in DNA and that this process is dependent on complementary binding of the crRNA to the target DNA. Two nuclease domains were found in Cas9 with the HNH domain cleaving the complementary strand and the RuvC-like domain cutting the non-complementary strand. Jinek et al. Science (2012) 337:816-821. http://www.ncbi.nlm.nih.gov/pubmed/22745249

2013: The CRISPR/Cas9 system was used to edit targeted genes in both human and mouse cells using designed crRNA sequences. Cong et al. Science (2013) 339:819-823. http://www.ncbi.nlm.nih.gov/pubmed/23287718

First use in plants. Li et al. Nat Biotechnol (2013) 8:688-691. http://www.ncbi.nlm.nih.gov/pubmed/23929339

Also first use in plants ? Nekrasov et al. Nat Biotechnol (2013) 8:691-693. http://www.ncbi.nlm.nih.gov/pubmed/23929340.

2014: The crystal structure of Cas9 complexed with both gRNA and targeted DNA was elucidated. Nishimasu et al. Cell (2014) 156:935-949. http://www.ncbi.nlm.nih.gov/pubmed/24529477

PAMs are identified as a key component of DNA target integration. Anders et al. Nature (2014) 513:569-573. http://www.ncbi.nlm.nih.gov/pubmed/25079318

sgRNA and Cas9 are directly delivered into cells without the use of a vector intermediate. Ramakrishna et al. Genome Res (2014) 24:1020-1027. http://www.ncbi.nlm.nih.gov/pubmed/24696462

2015: CRISPR/Cas9 was used to edit tri-chromosomal pre-implantation human embryos. Researchers attempted to repair the HBB locuswhich, when mutated, results in -thalassemia blood disorders. The researchers were unable to effectively repair the mutated locus and many off-target cleavages were observed. Liang et al. Protein and Cell (2015) 6:363-372. http://www.ncbi.nlm.nih.gov/pubmed/25894090

2015: An international moratorium is called for making heritable changes to the human genome using gene editing.At an international meeting convened by the National Academy of Science of the United States, the Institute of Medicine, The Chinese Academy of Sciences, and the Royal Society of London scientists called for a moratorium on making inheritable changes to the human genome. None of these groups have regulatory authority to prevent such research from taking place, however previous moratoriums where widely accepted in 1975 when an international group met in California to discuss gene editing in all species.http://www.nytimes.com/2015/12/04/science/crispr-cas9-human-genome-editing-moratorium.html?_r=0

2016: The USDA determines CRISPR/Cas9 edited crops will not be regulated as GMOs. Due to the lack of foreign DNA and the inability to distinguish CRISPR modified crops from those created by traditional plant breeding the USDA has determined that gene edited crops will not be regulated like traditional GMOs.http://www.nature.com/news/gene-edited-crispr-mushroom-escapes-us-regulation-1.19754

2016: The first human trial to use CRISPR gene editing gets approval from the NIH. A National Institute of Health advisory committee approved the use of CRISPR/Cas9 gene editing in a cancer therapy trial. The treatment will use CRISPR/Cas9 technology to edit the patients own T cells to target cancer.http://www.nature.com/news/first-crispr-clinical-trial-gets-green-light-from-us-panel-1.20137

Read the original here:
CRISPR Timeline CRISPR Update

The CRISPR-Cas9 gene editing tool shows incredible promise in treating a wide range of diseases, from HIV to cancer. But the technology isn't without controversy, as the long term effects of cutting DNA in living organisms isn't fully known. Now, scientists from the Salk Institute have modified CRISPR to work without making any cuts, switching targeted genes on and off instead, and demonstrated its effectiveness by treating diabetes, muscular dystrophy and other diseases in mice.

The CRISPR gene-editing tool is one of the most important scientific breakthroughs in years, with the potential to reverse the effects of disease or even snip them out of the genome at the embryo stage. But as exciting as it is, a recent study found that the cut-and-paste method may introduce unintentional mutations into the genome, and although this study was later contested, safety remains a concern at this early stage in the technology.

"Although many studies have demonstrated that CRISPR-Cas9 can be applied as a powerful tool for gene therapy, there are growing concerns regarding unwanted mutations generated by the double-strand breaks through this technology," says Juan Carlos Izpisua Belmonte, senior author of the study. "We were able to get around that concern."

The Salk scientists adapted the regular CRISPR mechanism to influence gene activation without actually changing the DNA itself. The Cas9 enzyme normally does the cutting, so the team used a dead form of it called dCas9 that can still target genes but doesn't damage them. The active ingredients this time are transcriptional activation domains, which act like molecular switches to turn specific genes on or off. These are coupled to the dCas9, along with the usual guide RNAs that help them locate the desired section of DNA.

There's just one problem with this technique: normally the CRISPR system is loaded into a harmless virus called an adeno-associated virus (AAV), which carries the tool to the target. But the entire protein, consisting of dCas9, the switches and the guide RNAs, is too big to fit inside one of these AAVs. To work around that issue, the researchers split the protein into two, loading dCas9 into one virus and the switches and guide RNAs into another. The guide RNAs were tweaked to make sure both parts still ended up at the target together, and to make sure the gene was strongly activated.

To test how well the new technique worked, the researchers experimented with mice that had three different diseases kidney damage, type 1 diabetes and muscular dystrophy. In each case, the mice were treated with specialized CRISPR systems to increase the expression of certain genes, which would hopefully reverse the symptoms.

In the kidney-damaged mice, the team targeted two genes that play a role in kidney function. Sure enough, there was an increase in the levels of a protein linked to those genes, and kidney function improved. In the diabetic mice, the targeted genes were those that promote the growth of insulin-producing cells, and after treatment, the mice were found to have lower blood glucose levels. And finally, the treatment also worked to reverse the symptoms of muscular dystrophy.

After that promising start, further work is underway on the system. The researchers plan to try to apply the technique to other cell types to help treat other diseases, and conduct more safety tests before human trials can begin.

The research was published in the journal Cell.

See the rest here:
CRISPR breakthrough treats diseases like diabetes without ...

C

RISPR-Cas9 is complicated.

Thats why scientists, entrepreneurs, and journalists like me have spent the past few years reaching for metaphors to try to make the mechanics of the revolutionary genome-editing technology easier for laypeople to understand. In text and imagery, weve drawn parallels to everything from garage tools to divine interventions.

But it must be said: Some of these analogies are better than others. To compile the definitive ranking, I sat down with STATs senior science writer Sharon Begley, a wordsmith who has herself compared CRISPR to 1,000 monkeys editing a Word document and the kind of dog you can train to retrieve everything from Frisbees to slippers to a cold beer.

advertisement

Sharon and I evaluated each of the metaphors we found by considering these three questions: Is it creative? Is it clear? And is it accurate? Below, our rankings of CRISPR analogies, ordered from worst to best:

This is not how it works. This is not at all how it works.

We see where these marketers got started with their pun: Genetics researchers do indeed use the term knock out to refer to eliminating an existing gene in, say, a mouse.

But a blunt instrument like a boxing glove vastly undersells CRISPRs precision. It also suggests, wrongly, that CRISPRs powers extend to leaving genes bruised and battered. For these reasons, this ad wins the ignoble prize as the worst CRISPR metaphor we could track down.

The hand of God is a familiar trope to describe advances in biotech. Elucidating CRISPR this way is sinful.

If God were in the business of editing the genome, we expect that She would make fewer mistakes than CRISPR, which is known foroff-target effects. Were wondering, too, if the holy light emanating from the hand of a CRISPR-ing God is meant to imply that She is among those researchers interested in combining CRISPR with optogenetics?

Most damningly, though, this metaphor does nothing to explain how CRISPR actually works.

The framing of CRISPR as a method to remove ticking time bombs lurking within our DNA is true enough: Researchers do want to use the technology to take out genetic mutations that cause deadly diseases.

But this visual metaphor confuses the biology. The destructive power in DNA lies in the base pairs themselves, not in between them, where this red canister is placed. And again, this does nothing to shine light on CRISPRs mechanism of action.

We had high hopes for this analogy, which came courtesy of the National Institutes of Health. But alas, it mostly disappoints.

The idea, as we understand it, is that CRISPR-Cas9 acts to modify precisely the correct segments of DNA, similar to how a handyman uses a particular wrench to loosen or tighten a nut or bolt of a specific size and shape.

But were scratching our heads to come up with a real-life construction scenario where whats visualized here would actually happen.We get the sense that someone in pursuit of a fresh analogy came up with this one only after concluding that all the good analogies were already taken.

This analogy is so 2012. Sure, an eraser is a fine way to think about CRISPRs powers to delete. But that only goes halfway what about CRISPRs powers to add or replace? And it loses the physicality of CRISPR-Cas9s cutting action for no good reason. (In the interests of full disclosure, we must admit that STAT has used this one in the past. Apologies.)

The notion of CRISPR as a surgeons scalpel nicely captures its cutting action. But points are deducted for the suggestion that CRISPR is as precise as a surgeons tool must be.

We like the simple explanatory power of a plain-old pair of scissors to describe CRISPR-Cas9s cutting action. Its better than the scalpel metaphor at conveying the technology isablunt instrument. But points are deducted for not addressing CRISPRs powers to add or replace.

This analogy comes by way ofthe authority:Feng Zheng, the groundbreaking Massachusetts Institute of Technology scientist who helped create CRISPR-Cas9.

Zhengs comparison is a good one overall especially when he explains how it works with the song Twinkle Twinkle Little Star. But its still an imperfect one, because it implies greater precision than CRISPR actually allows.

To continue the analogy: If you use CRISPR to search for the and replace it with this, it would work as intended sometimes. But because CRISPR sometimes finds something it shouldnt, you might also wind up with jumbled words describing the study of the divine as thisology and a book of synonyms as athissaurus.

We really like this comparison, exemplified bywriter Aime Lutkins turn of phrase describing CRISPR assort of like organic matter Photoshop.

To be sure, youre not literally cutting anything, as CRISPR-Cas9 does, when you use the Adobe image editing software. But we saw explanatory power in the fact that Photoshop lets you make zoomed-in changes, down to the level of a single pixel just as CRISPR can make changes at the level of the As, Ts, Cs, and Gs that make up the genetic code.

And as anyone whos been victim of a bad Photoshop job knows, theres plenty of room for the tool to go awry.

Folks, we have a winner: A Swiss Army knife is the best analogy we found for what CRISPR can and cant do.

Like the other cutting instruments on our list, a Swiss Army knife gets points as a good visual because CRISPR-Cas9 literally cuts DNA. But a Swiss Army knife breaks out of the pack because it has different blades for different tasks comparable to CRISPRs ability to cut something out, introduce a single one-letter change, or make an insertion without a deletion. Swiss Army knives also strike the right middle ground between a precise cut and a blunt cut, a good way to think about CRISPRs capabilities.

And if thats not enough: Both CRISPR and Swiss Army knives have recently been at the center of heated legalfights over intellectual property.

Business Reporter

Rebecca covers the business of biopharma.

Originally posted here:
The best and worst analogies for CRISPR, ranked

In the last five years, biology has undergone a seismic shift as researchers around the globe have embraced a revolutionary technology called gene editing. It involves the precise cutting and pasting of DNA by specialized proteinsinspired by nature, engineered by researchers. These proteins come in three varieties, all known by their somewhat clumsy acronyms: ZFNs, TALENs, and CRISPRs. But its Crispr, with its elegant design and simple cell delivery, thats most captured the imagination of scientists. Theyre now using it to treat genetic diseases, grow climate-resilient crops, and develop designer materials, foods, and drugs.

So how does it work?

When people refer to Crispr, they're probably talking about Crispr-Cas9, a complex of enzymes and genetic guides that together finds and edits DNA. But Crispr on its own just stands for Clustered Regularly Interspaced Palindromic Repeatschunks of regularly recurring bits of DNA that arose as an ancient bacterial defense system against viral invasions.

Viruses work by taking over a cell, using its machinery to replicate until it bursts. So certain bacteria evolved a way to fight back. They deployed waves of DNA-cutting proteins to chop up any viral genes floating around. If the bacteria survived the attacks, they'd incorporate tiny snippets of virus DNA into their own genomeslike a mug shot of every foe theyd ever come across, so they could spot each one quicker in the future. To keep their genetic memory palace in order, they spaced out each bit of viral code (so-called guide RNAs) with those repetitive, palindromic sequences in between. It doesn't really matter that they read the same forward and backward; the important thing is that they helped file away genetic code from viral invaders past, far away from more essential genes.

And having them on file meant that the next time a virus returned, the bacteria could send out a more powerful weapon. They could equip Cas9a lumpy, clam-shaped DNA-cutting proteinwith a copy of that guide RNA, pulled straight out of storage. Like a molecular assassin, it would go out and snip anything that matched the genetic mug shot.

Thats what happens in the wild. But in the lab, scientists have harnessed this powerful Crispr system to do things other than fight off the flu. The first step is designing a guide RNA that can sniff out a particular block of code in any living cellsay, a genetic defect, or an undesirable plant trait. If that gene consists of a string of the bases A, A, T, G, C, scientists make a complementary strand of RNA: U, U, A, C, G. Then they inject this short sequence of RNA, along with Cas9, into the cell theyre trying to edit. The guide RNA forms a complex with Cas9; one end of the RNA forms a hairpin curve that keeps it stuck in the protein, while the other endthe business enddangles out to interact with any DNA it comes across.

Once in the cell's nucleus, the Crispr-Cas9 complex bumps along the genome, attaching every time it comes across a small sequence called PAM. This protospacer adjacent motif is just a few base pairs, but Cas9 needs it to grab onto the DNA. And by grabbing it, the protein is able to destabilize the adjacent sequence, unzipping just a little bit of the double helix. That allows the guide RNA to slip in and sniff around to see if it's a match. If not, they move on. But if every base pair lines up to the target sequence, the guide RNA triggers Cas9 to produce two pincer-like appendages, which cut the DNA in two.

The process can stop there, and simply take a gene out of commission. Or, scientists can add a bit of replacement DNAto repair a gene instead of knocking it out.

And they don't have to limit themselves to just Cas9. There's a whole bunch of proteins that can use an RNA guide. There's Cas3, which gobbles up DNA Pac-Man style. Scientists are using it to develop targeted antibiotics that can wipe out a strain of C. diff, while leaving your gut microbiome intact. And there's an enzyme called Cas13 that works with a guide that gloms onto RNA, not DNA. Called Sherlock, the system is being used to develop sensitive tests for viral infections. Researchers are working hard to add more implements to the Crispr toolkit, but at least right now, Cas9 is still the most widely used.

Crispr isnt perfect; sometimes the protein veers off course and makes cuts at unintended sites. So scientists are actively working on ways to minimize these off-target effects. And as it gets better, the ethical questions surrounding the technology are going to get a lot thornier. Hello, designer babies?! Figuring out where those lines get drawn is going to take more than science; it will require policymakers and the public coming to the table. Because pretty soon with Crispr, the question wont be can we do it, but should we?

Excerpt from:
How Does Crispr Gene Editing Work? | WIRED

The debate about Johnny Depps casting as Grindelwald in the Fantastic Beasts and Where To Find Them franchise rages on. A quick recap: Depp was cast as Grindelwald a few years ago, but it was kept under wraps, as his casting was part of a big twist at the end of the first film.

But last summer, just before Fantastic Beasts was released, Amber Heard, his now ex-wife, accused Depp of domestic abuse. Videos of Depp throwing bottles and glasses and shouting at Heard, photos of Heards bruises, and text exchanges between Heard and Depps manager, all made their way into the press. The couple later released a joint statement saying neither party had lied for financial gain and that there wasnever any intent of physical or emotional harm.

The latest developments come as the press for the second Fantastic Beasts film, The Crimes of Grindelwald kicks into gear. The title, and images of Depp in character, reignited a debate over whether someone accused of domestic abuse should be given such a high-profile role, especially in a progressive childrens franchise. Depp has continuously denied all allegations since the divorce as salacious false stories.

On Tuesday, in an interview with Entertainment Weekly, Fantastic Beasts director David Yates explicitly defended Depps character, calling the concerns of audiences a dead issue. Yates attempted to positively contrast the allegations against Depps with those made against Harvey Weinstein and Kevin Spacey.

Honestly, theres an issue at the moment where theres a lot of people being accused of things, he said.[But] with Johnny, it seems to me there was one person who took a pop at him and claimed something.

Its very different [to instances] where there are multiple accusers over many years that need to be examined and we need to reflect on our industry that allows that to roll on year in and year out. Johnny isnt in that category in any shape or form. So to me, it doesnt bear any more analysis. It's a dead issue.

Yates also seemed to find it relevant that hehas had no direct experience of Depp being abusive towards him. He added: I can only tell you about the man I see every day: Hes full of decency and kindness, and thats all I see. Whatever accusation was out there doesnt tally with the kind of human being Ive been working with.

Its an interesting departure from Yatess previous take on the issue, which emphasised Depps talent over his private actions, separating art from artist. The whole principal of casting the movie was go with the best actor, he said last year. In this business, its a weird old business. Youre brilliant one week, people are saying odd things the next, you go up and down. But no one takes away your pure talent. Johnny Depp is a real artist.

Its possible that after the widely commended decisions from studios and directors to have both Harvey Wenstein and Kevin Spacey removed from their cinematic projects, Yates has felt the need to change tack. Instead of saying that the personal actions of an actor are irrelevant to their professional roles an argument that Hollywood and the public seem to have moved beyond he tries to separate Depp from those men by emphasising the differences in the actions themselves.

In 2016, J K Rowling simply said of Depps casting,Im delighted. Hes done incredible things with that character, again ensuring the focus stayed solely on talent rather than Depps character as a colleague or friend.

Those comments cause some controversy, and in the days after, she tweeted a couple of cryptic quotes seemingly referencing the situation. First, Those only who can bear the truth will hear it. - Arthur Helps. Then, Arguments cannot be answered by personal abuse; there is no logic in slander, and falsehood, in the long run, defeats itself - R G Ingersoll. When a fan messaged her to say, We know you'd never support abuse, Rowling, whose first husband Jorge Arantes has admitted to acting abusively,replied Thank you x. Its impossible to say from these quotes what her position actually is. Is the truth she references a belief that Depp is not really an abuser? Or is she hinting that she would like to re-cast the role, or condemn Depps casting, but is contractually unable to?

Since then, shes been silent on the issue. Although Rowlings actual thoughts on the situation are unclear, supporters of Depp see these cryptic tweets, the casting, and her general lack of condemnation of Depp, as evidence that she supports him. If you look up the hashtags #JohnnyDeppIsInnocent and #JohnnyDeppIsMyGrindelwald, J K Rowling immediately appears as a related person and related search term, as so many Depp supporters are mentioning the two in the same breath either thanking J K Rowling for her support, or using her name to argue that Depp must be innocent if even J K Rowling herself thinks so. Some have even used the hashtag #JohnnyDeppIsJKRowlingsGrindelwald.

Domestic violence is by nature a more difficult crime to discuss that serial instances of documented workplace sexual harassment it happens at home, with few or no witness, to people in complex existing romantic relationships.

In Amber Heards own words, When it happens in your home, behind closed doors, with someone you love, its not as straightforward as if a stranger did this. Clearly, Hollywood still finds allegations of domestic violence easier to swallow than workplace harassment.

See the article here:
CRISPR: can gene-editing help nature cope with climate change?

Genome editing (also called gene editing) is a group of technologies that give scientists the ability to change an organism's DNA. These technologies allow genetic material to be added, removed, or altered at particular locations in the genome. Several approaches to genome editing have been developed. A recent one is known as CRISPR-Cas9, which is short for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9. The CRISPR-Cas9 system has generated a lot of excitement in the scientific community because it is faster, cheaper, more accurate, and more efficient than other existing genome editing methods.

CRISPR-Cas9 was adapted from a naturally occurring genome editing system in bacteria. The bacteria capture snippets of DNA from invading viruses and use them to create DNA segments known as CRISPR arrays. The CRISPR arrays allow the bacteria to "remember" the viruses (or closely related ones). If the viruses attack again, the bacteria produce RNA segments from the CRISPR arrays to target the viruses' DNA. The bacteria then use Cas9 or a similar enzyme to cut the DNA apart, which disables the virus.

The CRISPR-Cas9 system works similarly in the lab. Researchers create a small piece of RNA with a short"guide" sequence that attaches (binds) to a specific target sequence of DNA in a genome. The RNA also binds to the Cas9 enzyme. As in bacteria, the modified RNA is used to recognize the DNA sequence, and the Cas9 enzyme cuts the DNA at the targeted location. Although Cas9 is the enzyme that is used most often, other enzymes (for example Cpf1) can also be used. Once the DNA is cut, researchers use the cell's own DNA repair machinery to add or delete pieces of genetic material, or to make changes to the DNA by replacing an existing segment with a customized DNA sequence.

Genome editing is of great interest in the prevention and treatment of human diseases. Currently, most research on genome editing is done to understand diseases using cells and animal models. Scientists are still working to determine whether this approach is safe and effective for use in people. It is being explored in research on a wide variety of diseases, including single-gene disorders such as cystic fibrosis, hemophilia, and sickle cell disease. It also holds promise for the treatment and prevention of more complex diseases, such as cancer, heart disease, mental illness, and human immunodeficiency virus (HIV) infection.

Ethical concerns arise when genome editing, using technologies such as CRISPR-Cas9, is used to alter human genomes. Most of the changes introduced with genome editing are limited to somatic cells, which are cells other than egg and sperm cells. These changes affect only certain tissues and are not passed from one generation to the next. However, changes made to genes in egg or sperm cells (germline cells) or in the genes of an embryo could be passed to future generations. Germline cell and embryo genome editing bring up a number of ethical challenges, including whether it would be permissible to use this technology to enhance normal human traits (such as height or intelligence). Based on concerns about ethics and safety, germline cell and embryo genome editing are currently illegal in many countries.

Gupta RM, Musunuru K. Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9. J Clin Invest. 2014 Oct;124(10):4154-61. doi: 10.1172/JCI72992. Epub 2014 Oct 1. Review. PubMed: 25271723. Free full-text available from PubMed Central: PMC4191047.

Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014 Jun 5;157(6):1262-78. doi:10.1016/j.cell.2014.05.010. Review. PubMed: 24906146. Free full-text available from PubMed Central: PMC4343198.

Komor AC, Badran AH, Liu DR. CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes. Cell. 2017 Apr 20;169(3):559. doi:10.1016/j.cell.2017.04.005. PubMed: 28431253.

Lander ES. The Heroes of CRISPR. Cell. 2016 Jan 14;164(1-2):18-28. doi:10.1016/j.cell.2015.12.041. Review. PubMed: 26771483.

Visit link:
What are genome editing and CRISPR-Cas9?

Designer babies, the end of diseases, genetically modified humans that never age. Outrageous things that used to be science fiction are suddenly becoming reality. The only thing we know for sure is that things will change irreversibly.

Support us on Patreon so we can make more videos (and get cool stuff in return): https://www.patreon.com/Kurzgesagt?ty=h

Kurzgesagt merch here: http://bit.ly/1P1hQIH

Get the music of the video here:

soundcloud: http://bit.ly/2aRxNZdbandcamp: http://bit.ly/2berrSWhttp://www.epic-mountain.com

Thanks to Volker Henn, James Gurney and (prefers anonymity) for help with this video!

THANKS A LOT TO OUR LOVELY PATRONS FOR SUPPORTING US:

Jeffrey Schneider, Konstantin Kaganovich, Tom Leiser, Archie Castillo, Russell Eishard, Ben Kershaw, Marius Stollen, Henry Bowman, Ben Johns, Bogdan Radu, Sam Toland, Pierre Thalamy, Christopher Morgan, Rocks Arent People, Ross Devereux, Pascal Michaud, Derek DuBreuil, Sofia Quintero, Robert Swiniarski, Merkt Kzlrmak, Michelle Rowley, Andy Dong, Saphir Patel, Harris Rotto, Thomas Huzij, Ryan James Burke, NTRX, Chaz Lewis, Amir Resali, The War on Stupid, John Pestana, Lucien Delbert, iaDRM, Jacob Edwards, Lauritz Klaus, Jason Hunt, Marcus : ), Taylor Lau, Rhett H Eisenberg, Mr.Z, Jeremy Dumet, Fatman13, Kasturi Raghavan, Kousora, Rich Sekmistrz, Mozart Peter, Gaby Germanos, Andreas Hertle, Alena Vlachova, Zdravko aek

SOURCES AND FURTHER READING:

The best book we read about the topic: GMO Sapiens

https://goo.gl/NxFmk8

(affiliate link, we get a cut if buy the book!)

Good Overview by Wired:http://bit.ly/1DuM4zq

timeline of computer development:http://bit.ly/1VtiJ0N

Selective breeding: http://bit.ly/29GaPVS

DNA:http://bit.ly/1rQs8Yk

Radiation research:http://bit.ly/2ad6wT1

inserting DNA snippets into organisms:http://bit.ly/2apyqbj

First genetically modified animal:http://bit.ly/2abkfYO

First GM patent:http://bit.ly/2a5cCox

chemicals produced by GMOs:http://bit.ly/29UvTbhhttp://bit.ly/2abeHwUhttp://bit.ly/2a86sBy

Flavr Savr Tomato:http://bit.ly/29YPVwN

First Human Engineering:http://bit.ly/29ZTfsf

glowing fish:http://bit.ly/29UwuJU

CRISPR:http://go.nature.com/24Nhykm

HIV cut from cells and rats with CRISPR:http://go.nature.com/1RwR1xIhttp://ti.me/1TlADSi

first human CRISPR trials fighting cancer:http://go.nature.com/28PW40r

first human CRISPR trial approved by Chinese for August 2016:http://go.nature.com/29RYNnK

genetic diseases:http://go.nature.com/2a8f7ny

pregnancies with Down Syndrome terminated:http://bit.ly/2acVyvg( 1999 European study)

CRISPR and aging:http://bit.ly/2a3NYAVhttp://bit.ly/SuomTyhttp://go.nature.com/29WpDj1http://ti.me/1R7Vus9

Help us caption & translate this video!

http://www.youtube.com/timedtext_cs_p...

Link:
CRISPR - youtube.com

The acronyms might not be quite as catchy as CRISPR, but what new genetic tools dubbed REPAIR and ABE lack in whimsy they promise to make up in utility. These advances, unveiled last week, solve two of the problems hobbling CRISPR, the revolutionary genome-editing technique: that its idea of editing is often like 1,000 monkeys editing a Word document, and that making permanent changes to DNA might not be the best approach.

Together, the discoveries, described in separate studies, show that five years after scientists demonstrated that CRISPR can edit DNA, bioengineers are still racing to develop the most efficient, precise, versatile and therefore lucrative genome-editing tools possible.

One reason these are so exciting is, they show the CRISPR toolbox is still growing, said chemical engineer Gene Yeo of the University of California San Diego. There are going to be a lot more, and its not going to stop anytime soon. His lab has been working on one of the CRISPR advances but was not involved in either of the two new studies; its personally frustrating to get beaten, he added.

One discovery, led by biochemist David Liu of Harvard University, extends his 2016 invention of a way to change a single DNA letter, or base, on the 3-billion-letter-long human genome. Classic CRISPR cuts DNA with a molecular scissors and leaves the cell to repair the breach willy-nilly, introducing the problem of 1,000 monkeys editing away. In contrast, Lius base editor replaces the molecular scissors with something like a pencil wielded by an expert forger: It is an enzyme that literally rearranges atoms, cleanly and without collateral damage that the cell needs to fix.

Get Talking Points in your inbox:

An afternoon recap of the days most important business news, delivered weekdays.

As in classic CRISPR, this version finds its way to a target on the genome via a molecule that acts like a bloodhound. Attached to the bloodhound is the atom-rearranger, which in Lius 2016 version turned the DNA letter G into A. Thousands of genetic diseases arise because a gene has a G where it should have an A, so the edit might one day treat or prevent them.

But other inherited disorders need different alphabetical magic. Thats what Liu, postdoctoral fellows Nicole Gaudelli and Alexis Komor, and their colleagues report in a paper in Nature: Their new ABE (adenine base editor) can turn A into G. Attached to CRISPRs bloodhound molecule, ABE works at virtually any target site in genomic DNA, Liu said.

In tests so far, it changed DNA in more of the lab-grown human cells that it was slipped into than standard CRISPR.

About half of the 32,000 known disease-causing, single-letter mutations have one of the misspellings that ABE can fix, Liu said. They include sickle cell, Tay-Sachs, and cystic fibrosis, raising hopes that ABE could be used to treat these diseases, or (in early embryos) prevent them. In tests of cells growing in lab dishes, ABE reversed the mutation that causes hereditary hemochromatosis in about 30 percent of the cells, and changed another gene into a form that prevents sickle cell disease even in people who have its disease-causing mutation.

As with all forms of CRISPR, before ABE helps any patients, scientists will have to test whether its safe and effective. But having the molecular machine is a good start, said Liu, a cofounder of the CRISPR company Editas Medicine Inc., based in Cambridge. He and his colleagues have filed for patents on ABE.

Harvard biologist George Church, who tied for first in the race to make CRISPR work in human cells, called base editing especially interesting. Changing a single DNA letter, he said, means fewer worries about the editing enzyme [in classic CRISPR] later going rogue or silent. He also expects that crops with a single base change will not be designated as transgenic, reducing regulatory barriers to commercialization.

In a separate study, CRISPR pioneer Feng Zhang of Cambridges Broad Institute discovered along with his colleagues a new version of CRISPR that can edit RNA, DNAs friskier cousin. While DNA mostly sits sedately in cells and issues orders to make proteins that keep life living, RNA zips around the cell carrying out those orders, and then disappears. That makes RNA a tantalizing target: By editing the errant orders (RNA) rather than their issuer (DNA), scientists might be able to make temporary, reversible genetic edits, rather than CRISPRs permanent ones.

Editing DNA is hard to reverse, but once you stop providing the RNA-editing system, the changes will disappear over time, said Zhang, also a cofounder of Editas. That might make it possible to treat conditions where you dont need a permanent edit, such as when the immune system is in overdrive and causing inflammation.

To create what Zhang and his colleagues call REPAIR (RNA editing for programmable A to I [G] replacement), they fused an enzyme that binds to RNA with one that changes the RNA letter A (adenosine) to inosine, a molecule similar to the RNA letter G (guanosine), they report in Science.

In tests on human cells growing in the lab, REPAIR corrected misspellings in the RNA that was made by disease-causing DNA in this case, Fanconi anemia, an inherited and devastating bone marrow disease, or nephrogenic diabetes insipidus, a serious inborn kidney disease.

The furious race to improve CRISPR, via ABE or REPAIR or whatever comes next, Church said, is a reminder of how far CRISPR is from precise genome-editing in humans.

Excerpt from:
CRISPR gene editing gets new tools, and acronyms

Scientists have used a popular gene editing tool called CRISPR to snip out a tiny piece of DNA from one particular gene in a white button mushroom. The resulting mushroom doesn't brown when cut. Adam Fagen/Flickr hide caption

Scientists have used a popular gene editing tool called CRISPR to snip out a tiny piece of DNA from one particular gene in a white button mushroom. The resulting mushroom doesn't brown when cut.

There's a genetic technology that scientists are eager to apply to food, touting its possibilities for things like mushrooms that don't brown and pigs that are resistant to deadly diseases.

And food industry groups, still reeling from widespread protests against genetically engineered corn and soybeans (aka GMOs) that have made it difficult to get genetically engineered food to grocery store shelves, are looking to influence public opinion.

The technology is called Clustered Regularly Interspaced Short Palindromic Repeats, or CRISPR. It's a technique that Alison Van Eenennaam, an animal genetics professor at University of California, Davis, says can de-activate a gene. Or, as she puts it: "It's editing. It's like going into a Word document and basically replacing one letter, maybe that instead of 'wind,' you want it to say 'wine,' " she says.

Skeptics, like Dana Perls with the environmental group Friends of the Earth, say food companies are trying to distance themselves from terms like GMO and genetic engineering that have caused them trouble with consumers.

"These new gene editing technologies like CRISPR are genetic engineering. And if this is genetic engineering, then call it that," says Perls. She says these producers are just trying to pull the wool over consumers' eyes with a strong public relations push.

Dozens of crops and livestock developed with CRISPR technology are years from the market, though the U.S. Department of Agriculture already said it won't regulate CRISPR-developed products like other genetically engineered food, since no foreign genetic material is introduced in the process. The Food and Drug Administration will decide which new products are safe.

To get ahead of any criticism, a group of heavyweights in the food industry have joined forces to form the Coalition for Responsible Gene Editing in Agriculture, which is funded by members like the U.S. Pork Board, Monsanto, Syngenta and Bayer.

The board's CEO, Bill Even, says the food industry missed a chance to do this when the earlier wave of genetically engineered food made it to the market.

"There was never any conversation with consumers around what is this and what did it mean," he says. "Fast forward now today, there's a lot of debate around GMOs and food. The public rightly [is] ... interested in knowing what's in their food."

People don't often trust big companies, says Charlie Arnot, who leads the coalition and is the CEO of the Center for Food Integrity. But when it comes to CRISPR, there are three key strategies Arnot says will help get consumers on board.

First: CRISPR is not a secret.

"Those in technology have to be more transparent and be much more engaged in a public conversation and dialogue, in order to answer those questions, address the skepticism and ultimately result in earning consumer trust in what they're doing in gene editing," he says.

Second, the coalition wants to show that it shares the same values that shoppers do. So, its members are sponsoring and attending events like CRISPRcon to engage in public discussions about the technology and its potential animal welfare, societal and environmental benefits.

"If people trust you, science doesn't matter. If people don't trust you, science doesn't matter," Arnot says. "It only matters after you cross that trust threshold. So you really have to engage in that values-based dialogue to build trust, and then you're given the permission to introduce the science."

And that's the third strategy: These companies want consumers to know that CRISPR isn't like other forms of genetic engineering. CRISPR changes the way genes are expressed; it doesn't necessarily add genetic material from another species, although it can be used that way.

"That's going to be the path that will ultimately lead to greater trust," Arnot says. "If we shortcut that path, we run the risk of potentially having this significantly beneficial technology not be accepted."

But persuading consumers to buy into CRISPR will be an uphill battle for Arnot and other industry groups. Food and environmental advocacy groups already are asking questions about CRISPR, as well as raising concerns over tracing genetically edited food in the system and the potential lack of regulatory oversight.

This story comes to us from Harvest Public Media, a reporting collaboration that focuses on agriculture and food production. Kristofor Husted is based at member station KBIA in Columbia, Mo.

More here:
Amid GMO Strife, Food Industry Vies For Public Trust In ...

The CRISPR Pill made headlines with its implications in the fight against Superbugs.

But CRISPR technology originated from research into gene splicing and genetic editing capabilities. Since DNA is the fundamental building block of existence, what CRISPR claims it can do is bold and a little terrifying.

What are the real-world applications and implications of this biotech?

The above image comes from a YouTube video produced by the McGovern Institute for Brain Research at MIT from 2014. You can see that it involves gene splicing. One of the faculty members, Feng Zhang, led a team of researchers at MIT on the project, but many groups have looked into CRISPR-Cas9 biotech.

As early as 1993, researcher Francisco Mojica of the University of Alicante in Spain tinkered with CRISPR. Fun fact: the CRISPR DNA sequenceandCas-9 enzyme are a naturally occurring defense mechanism in various bacteriamost notably the kind that causes strep throat.

Yeswe derived gene editing biotech from that pestering, cold weather (but also any time weather because its bacteria based) illness.

But dont worry: the CRISPR-Cas9 strand operates similarly to bacteriophages.

It repeats a series of the same DNA sequences with unique sequences peppered in. These clusters became known as clustered regularly interspaced short palindromic repeats.

Though Ruud Jansen first used the term CRISPR in 2002, Mojica adopted the initialization throughout his research in discovering that CRISPR is basically an adaptive immune system.

This led others to tinker with the bacteria-based defense mechanism, as well. In 2005, Alexander Bolotin of the French National Institute for Agricultural Research discovered the unusual Cas-9 protein displaying nuclease activity. He specifically noted it in the Streptococcus thermophilus bacteria as opposed to other bacteria. Bolotin also discovered a PAM(protospacer adjacent motif) which allows for target recognition.

From there, a plethora of scientists and researchers began to experiment with CRISPR and Cas-9 DNA sequences.

Knowing how something came to be is all fine and good, but exactly how does CRISPR work? Simple: it acts similarly to how viruses do when they attack organisms human or otherwise. Copies of the attacking virus DNA are made with temporary RNA. Then, these copies attach themselves to the attacked organism, forcing replication. This is how viruses infect things and its also how bacteriophages work, too.

Since researchers can now harness the power of CRISPR-Cas9 (bacterias own natural defense system against infection) many believe they can utilize this against antibiotic resistant strains of bacteria.

So, DNA has four amino-acid bases: adenine (A), thymine (T), guanine (G), and cytosine (C).

DNA will target any sequence acting a bit off to forego any potential damage. However, the CRISPR-Cas9 system operates differently since it can read 20 bases in any sequence. This elf-eyes-esque sight allows for better tailoring to a specific gene. There is even an online tool you can use to design a target sequence and how the RNA should interact with it.

While the implications of this are monstrous, the real world applications do meet with a few stumbling blocks. One such block: the fact that the enzymes sometimes cut at the wrong place. Clearly, when it comes to gene editing, you want to be able to hit your mark every time.

While the research into gene editing biotech has come a very long way, genetically engineered babies are still a bit further down the roador are they? Researchers in Portland, Oregon successfully edited a human embryo in 2017.

However, this falls under the category of germlinecells or reproductive cells. While the editing of somatic (or non-reproductive) cells is generally not controversial, editing reproductive cells raises several ethical dilemmas.

Despite this moral hang-up, use of CRISPR-Cas9 gene editing tech is already underwayeven in robotics. Transcriptics robotic lab added this biotech to its list of services in 2015 in hopes to save time and money in the gene editing process. China instigated human trials in 2016, and we dont even need to mention the implications regarding infectious diseases like Malaria.

We may have far to go with genetic editing and the fight against superbugs and viruses. But, we have taken very necessary and BIG first steps.

With how far genetics has come in the last 30 years, we have to wonder how advancements in robotics and biotech will propel things even further.

See the original post here:
The Latest Guide to Understanding CRISPR-Cas9

T

he acronyms might not be quite as catchy as CRISPR since, really, what is? but what new genetic tools dubbed REPAIR and ABE lack in whimsy they promise to make up in utility. These advances announced Wednesday solve two of the problems hobbling CRISPR, the revolutionary genome-editing technique: that its idea of editing is often like 1,000 monkeys editing a Word document, and that making permanent changes to DNA might not be the best approach.

Together, the discoveries, described in separate studies, show that five years after scientists demonstratedthat CRISPR can edit DNA, bioengineers are still racing to develop the most efficient, precise, versatile and therefore lucrative genome-editing tools possible.

One reason these are so exciting is, they show the CRISPR toolbox is still growing, said chemical engineer Gene Yeo of the University of California, San Diego. There are going to be a lot more, and its not going to stop anytime soon. His lab has been working on one of the CRISPR advances but was not involved in either of the two new studies its personally frustrating to get beaten, he added.

advertisement

One discovery, led by biochemist David Liu of Harvard University, extends his 2016 inventionof a way to change a single DNA letter, or base, on the 3-billion-letter-long human genome. Classic CRISPR cuts DNA with a molecular scissors and leaves the cell to repair the breach willy nilly, introducing the problem of 1,000 monkeys editing away. In contrast, Liusbase editor replaces the molecular scissors with something like a pencil wielded by an expert forger: It is an enzyme that literally rearranges atoms cleanly and without collateral damage that the cell needs to fix.

As in classic CRISPR, this version finds its way to a target on the genome via a molecule that acts like a bloodhound. Attached to the bloodhound is the atom-rearranger, which in Lius 2016 version turned the DNA letterG into A. Thousands of genetic diseases arise because a gene has a G where it should have an A,so the edit might one day treat or prevent them.

But other inherited disorders need different alphabetical magic. Thats what Liu, postdoctoral fellows Nicole Gaudelli and Alexis Komor, and their colleagues report in a paper in Nature: Their new ABE (adenine base editor) can turn A into G.Attached to CRISPRs bloodhound molecule, ABE works at virtually any target site in genomic DNA, Liu said.

In tests so far, it changed DNA in more of the lab-grown human cells thatit was slipped into than standard CRISPR (for all its fame, CRISPR often bungles the job). ABE also seems to make fewer off-target edits: In one test, it mistakenly hit four of the 12 off-target sites, compared to CRISPRs nine, and made that mistake in 1.3 percent of cases compared to 14 percent for CRISPR, Liu said.

About half of the 32,000 known disease-causing, single-letter mutations have one of the misspellings that ABE can fix, Liu said. They include sickle cell, Tay-Sachs, and cystic fibrosis, raising hopes that ABE could be used to treat these diseases, or (in early embryos) prevent them.In tests of cells growing in lab dishes, ABE reversed the mutation that causes hereditary hemochromatosis in about 30 percent of the cells, and changed another gene into a form that prevents sickle cell disease even in people who have its disease-causing mutation.

As with all forms of CRISPR, before ABE helps any patients, scientists will have to test whether its safe and effective. But having the molecular machine is a good start, said Liu, a co-founder of the CRISPR company Editas Medicine. He and colleagues have filed for patents on ABE.

Harvard biologist George Church, who tied for first in the race to make CRISPR work in human cells, called base editing especially interesting. Changing a single DNA letter, he said, means fewer worries about the editing enzyme [in classic CRISPR] later going rogue or silent. He also expects that crops with a single base change will not be designated as transgenic, reducing regulatory barriers to commercialization.

In a separate study, CRISPR pioneer Feng Zhang of the Broad Institute and his colleagues discovered a new version of CRISPR that can edit RNA, DNAs friskier cousin. While DNA mostly sits sedately in cells and issues orders to make proteins that keep life living, RNA zips around the cell carrying out those orders, and then disappears. That makes RNA a tantalizing target: By editing the errant orders (RNA) rather than their issuer (DNA), scientists might be able to make temporary, reversible genetic edits, rather than CRISPRs permanent ones.

Editing DNA is hard to reverse, but once you stop providing the RNA-editing system, the changes will disappear over time, said Zhang, also a co-founder of Editas. That might make it possible to treat conditions where you dont need a permanent edit, such as when the immune system is in overdrive and causing inflammation.

To create what Zhang and his colleagues call REPAIR (RNA editing for programmable A to I [G] replacement), they fused an enzyme that binds to RNA with one that changes the RNA letter A (adenosine) to inosine, a molecule similar tothe RNA letter G (guanosine), they report in Science.Other labs, including that of CRISPR developer Jennifer Doudna of the University of California, Berkeley, have also developed RNA editors, including one using the same Cas13 enzyme. But REPAIRs creators say theirs is more efficient and less error-prone.

In tests on human cells growing in the lab, REPAIR corrected misspellings in theRNA that was made by disease-causing DNA in this case, Fanconi anemia, an inherited and devastating bone marrow disease, or nephrogenic diabetes insipidus, a serious inborn kidney disease. Although the DNA still had its disease-causing mutations, 23 percent and 35 percent, respectively, of the RNA made by those defective genes was REPAIRed. Those levels might be high enough to treat the diseases. Some 5,800 inherited diseases are the result of the G-to-A glitch that REPAIR can fix, including epilepsy and Duchenne muscular dystrophy.

Both REPAIR and ABE might venture where CRISPR stumbles: in mature cells, like neurons, that dont divide. In unpublished research, Liu said, he and his team have shown that ABE can edit genes in neurons, raising the possibility of treating devastating neurological diseases with ABE.

The furious race to improve CRISPR, via ABE or REPAIR or whatever comes next, Church said, are potent reminders of how far CRISPR is from precise genome-editing in humans.

Senior Writer, Science and Discovery

Sharon covers science and discovery.

View original post here:
CRISPR toolbox gets two new molecular gadgets, boosting gene ...

Scientists have used a new gene-editing technique to create pigs that can keep their bodies warmer, burning more fat to produce leaner meat. Infrared pictures of 6-month-old pigs taken at zero, two, and four hours after cold exposure show that the pigs' thermoregulation was improved after insertion of the new gene. The modified pigs are on the right side of the images. Zheng et al. / PNAS hide caption

Scientists have used a new gene-editing technique to create pigs that can keep their bodies warmer, burning more fat to produce leaner meat. Infrared pictures of 6-month-old pigs taken at zero, two, and four hours after cold exposure show that the pigs' thermoregulation was improved after insertion of the new gene. The modified pigs are on the right side of the images.

Here's something that may sound like a contradiction in terms: low-fat pigs.

But that's exactly what Chinese scientists have created using new genetic engineering techniques.

In a paper published Monday in the Proceedings of the National Academy of Sciences, the scientists report that they have created 12 healthy pigs with about 24 percent less body fat than normal pigs.

The scientists created low-fat pigs in the hopes of providing pig farmers with animals that would be less expensive to raise and would suffer less in cold weather.

"This is a big issue for the pig industry," says Jianguo Zhao of the Institute of Zoology at the Chinese Academy of Sciences in Beijing, who led the research. "It's pretty exciting."

The genetically modified low-fat piglets Jianguo Zhao hide caption

The animals have less body fat because they have a gene that allows them to regulate their body temperatures better by burning fat. That could save farmers millions of dollars in heating and feeding costs, as well as prevent millions of piglets from suffering and dying in cold weather.

"They could maintain their body temperature much better, which means that they could survive better in the cold weather," Zhao said in an interview.

Other researchers call the advance significant.

"This is a paper that is technologically quite important," says R. Michael Roberts, a professor in the department of animal sciences at the University of Missouri, who edited the paper for the scientific journal. "It demonstrates a way that you can improve the welfare of animals at the same as also improving the product from those animals the meat."

But Roberts doubts the Food and Drug Administration would approve a genetically modified pig for sale in the United States. He's also skeptical that Americans would eat GMO pig meat.

"I very much doubt that this particular pig will ever be imported into the USA one thing and secondly, whether it would ever be allowed to enter the food chain," he says.

The FDA has approved a genetically modified salmon, but the approval took decades and has been met with intense opposition from environmental and food-safety groups.

Others say they hope genetically modified livestock will eventually become more acceptable to regulators and the public.

"The population of our planet is predicted to reach about 10 billion by 2050, and we need to use modern genetic approaches to help us increase the food supply to feed that growing population," says Chris Davies, an associate professor in the school of veterinary medicine at Utah State University in Logan, Utah.

Zhao says he doubts the genetic modification would affect the taste of meat from the pigs.

"Since the pig breed we used in this study is famous for the meat quality, we assumed that the genetic modifications will not affect the taste of the meat," he wrote in an email.

The Chinese scientists created the animals using a new gene-editing technique known as CRISPR-Cas9. It enables scientists to make changes in DNA much more easily and precisely than ever before.

Pigs lack a gene, called UCP1, which most other mammals have. The gene helps animals regulate their body temperatures in cold temperatures. The scientists edited a mouse version of the gene into pig cells. They then used those cells to create more than 2,553 cloned pig embryos.

Next, scientists implanted the genetically modified cloned pig embryos into 13 female pigs. Three of the female surrogate mother pigs became pregnant, producing 12 male piglets, the researchers report.

Tests on the piglets showed they were much better at regulating their body temperatures than normal pigs. They also had about 24 percent less fat on their bodies, the researchers report.

"People like to eat the pork with less fat but higher lean meat," Zhao says.

The animals were slaughtered when they were six months old so scientists could analyze their bodies. They seemed perfectly healthy and normal, Zhao says. At least one male even mated, producing healthy offspring, he says.

Read the original here:
CRISPR Bacon: Chinese Scientists Create Genetically Modified ...

Provided by The Verge

The powerful gene-editing tool CRISPR has been making headlines for its ability to edit DNA, which could one day transform how we fight cancer and other life-threatening diseases. Now, scientists have created a new version of CRISPR that can target and edit a different genetic building block: RNA.

The new tool, described in a study published today in Science, offers several advantages: its edits, for instance, arent permanent, which makes gene editing much safer. Researchers showed that the new system, called REPAIR, can work relatively efficiently in human cells. In the future, it could be used to treat diseases, as well as better understand the role that RNA plays in causing those diseases.

Its another tool in the toolbox.

Its another tool in the toolbox that we didnt have access to before, says Mitchell O'Connell, assistant professor in the Department of Biochemistry and Biophysics at the University of Rochester, who was not involved in the research. Its like developing new technology that makes you see things that you couldn't see before, or tweak things that you couldn't tweak.

The gene-editing tool CRISPR is based on a defense mechanism bacteria use to ward off viruses by cutting off bits of their DNA and pasting them elsewhere. Scientists have engineered that mechanism to tweak DNA, creating unusually muscular beagles, for instance, and mosquitoes that dont transmit malaria. But there are different types of CRISPR, with different types of molecular scissors. The gene-editing tool thats been making lots of headlines is called CRISPR-Cas9. The CRISPR used in todays study is called CRISPR-Cas13.

Instead of snipping DNA, this type of CRISPR targets another of the major biological molecules found in all forms of life, RNA. Most of the time, RNA is used inside the body to help DNA build proteins. And proteins play an important role in causing diseases. There are some advantages to editing RNA instead of DNA, says study co-author David Cox, a PhD student in the Zhang Lab at the Broad Institute of MIT and Harvard, which has been doing pioneering work on CRISPR. RNA is constantly being made and recycled inside cells, so an RNA edit is not permanent. (The edits could still be effective, though: the CRISPR system could be kept in cells for, say, months, allowing the scissors to keep editing RNA as it forms.) That makes the whole process safer. If you edit RNA and make a mistake, for instance, the faulty RNA will be degraded likely within 24 hours. Instead, if you edit DNA and make a mistake, that mistake is irreversible and could possible lead to cancer. Certain changes to DNA could also be passed on to future generations, while changes to RNA generally arent passed on.

there are still big risks involved.

Gene editing is very exciting, but there are still big risks involved, O'Connell tells The Verge. Targeting RNA rather than DNA is a safer strategy, particularly for things where you might not want to make permanent change.

To create the new editing tool, called REPAIR, the researchers combined CRISPR-Cas13 with a protein called ADAR. It works this way: the Cas13 enzyme is programmed to target a specific RNA sequence that might correspond to a disease mutation; the ADAR protein then makes the edit. In the study, the researchers showed that the system can edit specific RNA bases with 20 to 40 percent efficiency and up to 90 percent in some instances, says Cox. And the system made few mistakes: even though gene-editing tools are very precise, sometimes they snip pieces of genetic code they werent programmed to cut. These off-target cuts can be dangerous, and scientists want to make sure there are as few of them as possible.

A first version of REPAIR caused nearly 20,000 off-target cuts, says study co-author Omar Abudayyeh, also a PhD student in the Zhang Lab. That was a pretty disappointing moment, he tells The Verge. But then, the team tweaked the system in a way that reduced the number of off-target cuts to 10 to 20 per target site, making it much more precise and safe.

O'Connell says he was surprised by how well the system works. RNA has been targeted before in an effort to make drugs to treat disease, O'Connell says. But this CRISPR system makes the whole editing process much easier. In the future, this editing tool could be used to treat life-threatening diseases like hemophilia, as well as a heart condition called hypertrophic cardiomyopathy, which can lead to sudden death, Cox tells The Verge. Before that happens, the system needs to be optimized, and made much more precise. Researchers also need to show that it works in mice, other animals, and eventually in people. Its a long road to translate this into any sort of therapy, says study co-author Jonathan Gootenberg, another PhD student in the Zhang Lab.

You feel so empowered where youre in the lab.

Together with CRISPR-Cas9, this system really has the potential to revolutionize how we treat diseases. And thats the motivation that keeps Gootenberg, Cox, and Abudayyeh working hard in their lab. Abudayyeh says that when he was in med school, he met a woman with terminal lung cancer, who had maybe a few more months to live. You feel pretty hopeless in that situation because theres nothing you can do even as a doctor, he says. But thats also what inspired him to get into biotechnology.

You feel so empowered where youre in the lab, just thinking about new ways to make new technologies with the potential to hopefully actually help patients like that, Abudayyeh says. Its really exciting.

See the article here:
Gene-editing tool CRISPR can now manipulate more types of ...

Photo: LOREN ELLIOTT, Special To The Chronicle

A heat map illustrates how effectively mutated cells blocked HIV, in a UCSF lab.

A heat map illustrates how effectively mutated cells blocked HIV, in a UCSF lab.

How CRISPR gene-editing tech can fight HIV

Researchers at UCSF have received a three-year, $1.6 million grant to advance their work using novel gene-editing technology to make human blood cells less susceptible to HIV infection.

The grant, from biopharmaceutical giant Gilead Sciences, a global leader in sales of HIV treatments, will fund a team of scientists working to modify the DNA of a type of white blood cell to make them immune to HIV infection.

The cells, called T cells, have long been a focus of researchers seeking to improve HIV treatments. T cells help the immune system fight many diseases, including some cancers and flu viruses. They play a unique role in HIV because the virus targets and destroys T cells, and HIV-positive patients whose T cells become too depleted by the virus will progress to AIDS.

Using a gene-editing technique known as CRISPR, the UCSF researchers have already tested dozens of genes believed to play a role in how HIV spreads within the body. They do this by collecting blood samples from HIV-negative patients, altering the DNA of those cells, and then introducing the HIV virus to the modified cells in test tubes. Within two weeks, they can see whether the change to the gene has eliminated the cells ability to become infected with HIV.

CRISPR can be used to modify the DNA of plants, animals and other living organisms. It is considered a groundbreaking method because it is simpler and cheaper than other gene-editing techniques.

This is connecting CRISPR to HIV and opening up whole new avenues of research in understanding the interplay between human genetics and HIV, said Alex Marson, an assistant professor of microbiology and immunology at UCSF who leads the lab that received the Gilead grant.

The grant, announced this week, will allow Marsons lab to pursue an ambitious goal of uncovering why HIV remains dormant in some cells, only to awaken unpredictably, sometimes years later. Known as HIV latency, this characteristic of the virus is why HIV-positive patients must take antiretroviral drugs which are only effective in attacking the awake HIV for life.

The tricky thing about HIV, and one reason its so hard to cure, is that it can hide in the DNA of the human cells, said Joe Hiatt, a doctoral student of medicine and philosophy in Marsons lab and a leader in the research initiative. It becomes DNA and integrates into your DNA.

The problem has perplexed researchers for years. But Marson and Hiatt see potential for using CRISPR to discover which genes control HIV latency. They hope to use the gene-editing tool to create latent HIV cells in test tubes, and then modify the DNA in those cells to see which edits may coax the HIV out of hiding and make it susceptible to drugs. This will be the most challenging and complicated part of the research. If done successfully, it could lead to the development of drugs that target latent HIV and perhaps cure HIV permanently.

CRISPR technology is potentially revolutionary because HIV is a type of virus that will sneak its own genetic code into the genetic code of the human cell, said Ross Wilson, a scientist at UC Berkeleys Innovative Genomics Institute who is not involved in the grant. Its like hiding a book in a stack at the library, and the book has instructions to build a nasty bomb. To get rid of that information, you need to get it back out of the library. Weve never had the technology to do that inside the living cell until CRISPR came along. Its the first efficient way to do that inside living cells.

It is the first research initiative that Foster Citys Gilead, through its philanthropic program, has funded that involves using CRISPR as a tool in HIV cure-related research. While $1.6 million is not a huge amount, it comes with fewer restrictions than many government grants. The grant will fund a team of five researchers for three years.

It is one of five grants totaling $7.5 million, announced this week, that Gilead has awarded research institutions for HIV and AIDS-related initiatives. The others are to the University of Massachusetts Medical School; Dana-Farber Cancer Institute; Institute of Human Genetics, French National Center for Scientific Research and University of Montpellier; and Frederick National Laboratory for Cancer Research, AIDS and Cancer Virus Program.

A Gilead spokesman said that if the UCSF researchers discover how latent HIV can be targeted by drugs, the company will not necessarily have rights to licensing agreements or other commercial benefits. The grant is from the companys philanthropy program and is meant to support HIV research independent of Gileads business interests, he said.

Catherine Ho is a San Francisco Chronicle staff writer. Email: cho@sfchronicle.com Twitter: @Cat_Ho

Read more:
How CRISPR gene-editing tech can fight HIV - SFGate

Photos from Eadweard Muybridges study of a galloping horse have been recorded in bacterial DNA

Eadweard Muybridge/The LIFE Picture Collection/Gett

By Douglas Heaven

Life is an open book and were writing in it. A team at Harvard University has used the CRISPR genome-editing tool to encode video into live bacteria demonstrating for the first time that we can turn microbes into librarians that can pass records on to their descendants and perhaps to ours.

The technique could even let us create populations of cells that keep their own event logs, making records as biological processes like disease or brain development happen.

DNA is one of the best media for storing data we know of. Researchers have already crammed large amounts of information from books to digital images into tiny amounts of biological material. In theory, a gram of single-stranded DNA can encode 455 exabytes, or roughly 100 billion DVDs.

Advertisement

Most previous DNA storage work has used artificial DNA: digital information is translated into a DNA sequence that is then synthesized.

However, using CRISPR lets you cut and paste the digital information directly into the DNA of a live organism, in this case a large population of E. coli.

Bacteria use the CRISPR/Cas9 system to record information in their DNA about viruses they encounter. And this machinery has been co-opted by researchers to enable us to precisely edit genomes.

In bacteria, each new entry gets stored upstream of the last one, which makes it possible to read off a history of events in the order they happened. Previous groups have created lifelogging cells by using CRISPR/Cas9 to mark the genome when a particular event occurs. But these marks just provide a tally of how many times something happens.

Seth Shipman at Harvard University and his colleagues have now used a version of CRISPR with a different enzyme, called CRISPR/Cas1-Cas2. This let them add a message to the genome rather than simply cut a notch.

The message was a recorded image of a human hand and five images showing a galloping horse, taken from Eadweard Muybridges 1878 photographic study of the animals motion, which has since been animated.

Seth Shipman

To get the DNA sequences encoding this data inside the cells, the team applied an electrical current that opened channels in the cells walls and the DNA flowed in. Once inside, CRISPR got to work.

To read the data back again, the team sequenced the DNA of more than 600,000 cells. The large number is necessary because most cells will not have edited their genome entirely accurately. Every cell isnt going to acquire every piece of information we throw at it, says Shipman. The more cells that are sampled, the better the reconstruction of the data. Fortunately, with modern sequencing tools, reconstruction is quick.

The five frames of a horse in motion showed that it is possible to capture data chronologically and replay them as a video. You get a physical record of events over time, says Shipman. For a long time we wanted to have some way of storing timing information inside cells, says Shipman. The CRISPR system is perfectly adapted to that.

This is a really neat paper, says Yaniv Erlich at Columbia University in New York. The team didnt store that much data and it is not clear that the CRISPR technique can compete with the storage capacity of synthetic DNA. But inserting information into living cells opens up a lot of possibilities, he says.

For a start, it lets you add to or change the stored information later. And because the data is written into the bacterial genomes, it gets passed down between generations. Mutations happen, but not nearly as many as you think, says Shipman certainly not enough to corrupt the data stored across a large population of cells.

Storing data in bacteria could even be a way to make important information survive a nuclear apocalypse. You could useDeinococcus radiodurans, a species that maintains its genome in extreme radiation conditions, says Erlich.

Shipman wants to turn cells into recording devices that document what takes place inside themselves. He is excited about the possibility of keeping a log book of events inside a living brain as it develops, showing how different brain cells acquire their distinct identities.

Its hard to understand what events make brain cells fully defined, says Shipman. You cant easily get in there to take a look. Taking a brain apart disrupts the whole process.

You could also get a cell to diarise what happens as it changes from healthy to diseased. Now that would be an account worth reading.

Journal reference: Nature, DOI: 10.1038/nature23017

More on these topics:

The rest is here:
Video stored in live bacterial genome using CRISPR gene ...

CRISPR, a new genome editing tool, could transform the field of biologyand a recent study on genetically-engineered human embryos has converted this promise into media hype. But scientists have been tinkering with genomes for decades. Why is CRISPR suddenly such a big deal?

The short answer is that CRISPR allows scientists to edit genomes with unprecedented precision, efficiency, and flexibility. The past few years have seen a flurry of firsts with CRISPR, from creating monkeys with targeted mutations to preventing HIV infection in human cells. Earlier this month, Chinese scientists announced they applied the technique to nonviable human embryos, hinting at CRISPRs potential to cure any genetic disease. And yes, it might even lead to designer babies. (Though, as the results of that study show, its still far from ready for the doctors office.)

In short, CRISPR is far better than older techniques for gene splicing and editing. And you know what? Scientists didnt invent it.

CRISPR is actually a naturally-occurring, ancient defense mechanism found in a wide range of bacteria. As far as back the 1980s, scientists observed a strange pattern in some bacterial genomes. One DNA sequence would be repeated over and over again, with unique sequences in between the repeats. They called this odd configuration clustered regularly interspaced short palindromic repeats, or CRISPR.

This was all puzzling until scientists realized the unique sequences in between the repeats matched the DNA of virusesspecifically viruses that prey on bacteria. It turns out CRISPR is one part of the bacterias immune system, which keeps bits of dangerous viruses around so it can recognize and defend against those viruses next time they attack. The second part of the defense mechanism is a set of enzymes called Cas (CRISPR-associated proteins), which can precisely snip DNA and slice the hell out of invading viruses. Conveniently, the genes that encode for Cas are always sitting somewhere near the CRISPR sequences.

Here is how they work together to disable viruses, as Carl Zimmer elegantly explains in Quanta:

As the CRISPR region fills with virus DNA, it becomes a molecular most-wanted gallery, representing the enemies the microbe has encountered. The microbe can then use this viral DNA to turn Cas enzymes into precision-guided weapons. The microbe copies the genetic material in each spacer into an RNA molecule. Cas enzymes then take up one of the RNA molecules and cradle it. Together, the viral RNA and the Cas enzymes drift through the cell. If they encounter genetic material from a virus that matches the CRISPR RNA, the RNA latches on tightly. The Cas enzymes then chop the DNA in two, preventing the virus from replicating.

There are a number Cas enzymes, but the best known is called Cas9. It comes from Streptococcus pyogenes, better known as the bacteria that causes strep throat. Together, they form the CRISPR/Cas9 system, though its often shortened to just CRISPR.

Top image: Screenshot from this MIT video explaining CRISPR

As this point, you can start connecting the dots: Cas9 is an enzyme that snips DNA, and CRISPR is a collection of DNA sequences that tells Cas9 exactly where to snip. All biologists have to do is feed Cas9 the right sequence, called a guide RNA, and boom, you can cut and paste bits of DNA sequence into the genome wherever you want.

DNA is a very long string of four different bases: A, T, C, and G. Other enzymes used in molecular biology might make a cut every time they see, say, a TCGA sequence, going wild and dicing up the entire genome. The CRISPR/Cas9 system doesnt do that.

Cas9 can recognize a sequence about 20 bases long, so it can be better tailored to a specific gene. All you have to do is design a target sequence using an online tool and order the guide RNA to match. It takes no longer than few days for the guide sequence to arrive by mail. You can even repair a faulty gene by cutting out it with CRISPR/Cas9 and injecting a normal copy of it into a cell. Occasionally, though, the enzyme still cuts in the wrong place, which is one of the stumbling blocks for wider use, especially in the clinic.

Mice whose genes have been altered or knocked out (disabled) are the workhorses for biomedical research. It can take over a year to establish new lines of genetically-altered mice with traditional techniques. But it takes just few months with CRISPR/Cas9, sparing the lives of many mice and saving time.

Traditionally, a knockout mouse is made using embryonic stem (ES) cells. Researchers inject the altered DNA sequence into mouse embryos, and hope they are incorporated through a rare process called homologous recombination. Some of first generation mice will be chimeras, their bodies a mixture of cells with and without the mutated sequence. Only some of the chimeras will have reproductive organs that make sperm with mutated sequence. Researchers breed those chimeras with normal mice to get a second generation, and hope that some of them are heterozygous, aka carrying one normal copy of the gene and one mutated copy of the gene in every cell. If you breed two of those heterozygous mice together, youll be lucky to get a third generation mouse with two copies of the mutant gene. So it takes at least three generations of mice to get your experimental mutant for research. Here it is summarized in a timeline:

But heres how a knockout mouse is made with CRISPR. Researchers inject the CRISPR/Cas9 sequences into mouse embryos. The system edits both copies of a gene at the same time, and you get the mouse in one generation. With CRISPR/Cas9, you can also alter, say, fives genes at once, whereas you would have to had to go that same laborious, multi-generational process five times before.

CRISPR is also more efficient than two other genome engineering techniques called zinc finger nuclease (ZFN) and transcription activator-like effector nucleases (TALENs). ZFN and TALENs can recognize longer DNA sequences and they theoretically have better specificity than CRISPR/Cas9, but they also have a major downside. Scientists have to create a custom-designed ZFN or TALEN protein each time, and they often have to create several variations before finding one that works. Its far easier to create a RNA guide sequence for CRISPR/Cas9, and its far more likely to work.

Most science experiments are done on a limited set of model organisms: mice, rats, zebrafish, fruit flies, and a nematode called C. elegans. Thats mostly because these are the organisms scientists have studied most closely and know how to manipulate genetically.

But with CRISPR/Cas9, its theoretically possible to modify the genomes of any animal under the sun. That includes humans. CRISPR could one day hold the cure to any number of genetic diseases, but of course human genetic manipulation is ethically fraught and still far from becoming routine.

Closer to reality are other genetically modified creaturesand not just the ones in labs. CRISPR could become a major force in ecology and conservation, especially when paired with other molecular biology tools. It could, for example, be used to introduce genes that slowly kill off the mosquitos spreading malaria. Or genes that put the brakes on invasive species like weeds. It could be the next great leap in conserving or enhancing our environmentopening up a whole new box of risks and rewards.

With the recent human embryo editing news, CRISPR has been getting a lot of coverage as a future medical treatment. But focusing on medicine alone is narrow-minded. Precise genome engineering has the potential to alter not just us, but the entire world and all its ecosystems.

More Reading:

Breakthrough DNA Editor Borne of Bacteria Quanta, Carl Zimmer

A CRISPR For-CAS-t The Scientist, Carina Storrs

Genetically Engineering Almost Anything NOVA NEXT, Tim De Chant and Eleanor Nelsen

This post has been updated to clarify that the the number of basepairs in guide RNA for CRISPR/Cas9 is different from the number of basepairs it recognizes in a target sequence.

Contact the author at sarah@gizmodo.com.

Read the original post:
Everything You Need to Know About CRISPR, the New Tool that ...

Agriculture has come a long way in the past century. We produce more food than ever before but our current model is unsustainable, and as the worlds population rapidly approaches the 8 billion mark, modern food production methods will need a radical transformation if theyre going to keep up. But luckily, theres a range of new technologies that might make it possible. In this series, well explore some of the innovative new solutions that farmers, scientists, and entrepreneurs are working on to make sure that nobody goes hungry in our increasingly crowded world.

Corn isnt the sexiest crop but its one of the most important. Its the most abundant grain on Earth, used as food and biofuel around the globe. In ancient times, Mesoamericans thrived on it, waged wars over it. Their myths claimed corn was the matter from which gods created mankind itself.

But, just as corn helped create these civilizations, these civilizations helped create corn through meticulous selective breeding. Todays grain hardly resembles its ancestors. Compared to the wild plant first cultivated by ancient Mexicans some ten thousand years ago, modern corn is a super mutant.

And yet, after all those thousands of years of cultivation, just two main genes are thought to be responsible for the evolution of the corn we eat today. Selective breeding is painstakingly slow and imprecise.

But thats all about to change.

Selective breeding is painstakingly slow and imprecise. But thats all about to change.

New gene editing tools like CRISPR/Cas9 now let scientists hack into genomes, make precise incisions, and insert desired traits into plants and animals. Well soon have corn with higher crop yields, mushrooms that dont brown, pigs with more meat on the bone, and disease resistant cattle. Changes that took years, decades, or even centuries, can now be made in a matter of months. In the next five years you might eat tortilla chips made from edited corn. By 2020 you might drink milk from an edited cow.

Dubbed the CRISPR Revolution these scientific advances in gene editing have huge potential that many experts think could help fortify our food system and feed an increasing population of farmers who are threatened by food scarcity caused, in part, by climate change.

But not everyone is so certain. Beyond the contentious legal battles that have thus far complicated CRISPR science, calling into question who can and cant use the technology, some consumer rights advocates think these tools will be used to maintain the status quo of an industry based primarily on corporate profit. Meanwhile, residual worry about genetically modified organisms (GMOs) may influence the public perception of gene-edited organisms, steering consumers towards the organic aisle despite scientific evidence.

Gene editing is, simply put, the act of making intentional changes to DNA in order to create an organism with a specific trait or traits. Its like using a word processor to edit the words in a sentence. Geneticists insist we dont confuse this with genetic modification (otherwise called genetic engineering), which introduces new genes from different species in order to achieve desired traits. The difference may sound trivial but experts say it could help calm the concerns associated with GMOs.

Consider this simplification. We have the sentence, The cat has a hat, but want to be more descriptive about the hats color. With modification, we would borrow the German word for black and write, The cat has a schwarz hat. The sentence makes sense (sort of) but its obvious that to some people it would be problematic and maybe even an improper use of language. With editing, we dont have to borrow a word from another language. We instead just insert the English word and write, The cat has a black hat.

In the older, more traditional system, scientists were taking a gene from one species and putting it into a plant to confer a particular trait on that plant, Rachel Haurwitz, co-founder of Caribou Biosciences, told Digital Trends. Thats not what were looking to do. Were looking to use CRISPR gene editing to achieve the same outcome as we can get from traditional breeding, just faster.

This ability to edit with such speed and precision is still relatively new, and due largely to CRISPR, which emerged straight from nature to become the most popular and powerful gene editing tool used today. Discovered in bacteria in the late eighties, it wasnt until 2005 that researchers began to unravel its role. Scientists found that when certain bacteria come under attack from viruses, they use special enzymes to cut, copy, and save a bit of the viral DNA. Later, if the intruder returns, the bacteria can quickly recognize it and react to defend itself.

A few years later, researchers realized this system could be used to cut and edit the DNA of any organism, not just viruses. In 2012, Jennifer Doudna and Emmanuelle Charpentier published the first paper demonstrating how CRISPR can be used to edit an organisms genome.

Were looking to use CRISPR gene editing to achieve the same outcome as we can get from traditional breeding, just faster.

Not only is this technique far cheaper, faster, and more precise than conventional genetic modification, it avoids many (if not all) of the issues raised by skeptics, whose main concerns point toward the creation of transgenic organisms.

But, whereas genetic modification entails combining DNA from multiple species, gene editing entails altering the DNA of one species with a trait that already exists naturally.

Gene editing is not at all about taking DNA from a foreign species and integrating it into a plant, Haurwitz said. Its really about working within the constraints of the plants own genome.

Just over four years ago, Haurwitz founded Caribou as a spin off from Doudnas lab at the University of California, Berkeley. Since then, her team has partnered with companies around the world, providing licensing rights to use the startups version of the gene editing tool. One of those partnerships may see the first CRISPR-edited organism come to market via DuPont Pioneer, one of the worlds biggest chemical companies.

The day before Halloween 2015, Yinong Yang submitted an Am I Regulated letter to the United States Department of Agricultures (USDA) Animal and Plant Health Inspection Service (APHIS). He and his colleagues at Penn State had used CRISPR to knock out a gene in white button mushrooms that makes them go brown over time. Without the browning gene, white buttons look better and last longer, and Yang wanted to know whether his mushrooms could legally go to market.

The following spring, the departments response resonated throughout the scientific and agricultural community. APHIS does not consider CRISPR/Cas9-edited white button mushroomsto be regulated, it wrote in an open letter.

Last year, researchers at DuPont Pioneer, the agriculture branch of the multi-billion-dollar conglomerate DuPont, published a study about a strain of corn engineered with CRISPR to be more resistant to drought. Its one of several CRISPR-modified crops that may soon be coming to market.

It was a landmark decision. Yangs mushrooms were the first gene-edited crop cleared for commercial sale by the USDA, which made a clear distinction between genetic modification and gene editing, and set a precedent for those to come.

A few days later, DuPont the fourth largest chemical corporation in the world received a similar response from the USDA regarding its CRISPR-edited waxy corn thats disease resistant and drought tolerant. DuPont wasted no time announcing plans to take its crop to market within the next five to ten years.

The USDA has said these products do not fall into their remit, as their remit is really focused on, say, plant pathogens or noxious weeds, said Haurwitz, whose company provides DuPont with its CRISPR technology. At the same time were seeing the FDA put out a call for information as theyre looking at their own remit to oversee the entire food supply, not just products made with modern biotechnology. And I think theyre looking to members of the scientific and business communities to really weigh in over the next few months.

Unlike most Button mushrooms, these ones dont brown or develop blemishes from being handled. This trait doesnt occur naturally it happens because the gene that makes the mushrooms turn brown was selectively removed from them via the CRISPR/Cas9 method. (Photo: Yang Lab)

For Yangs part, he intends to improve his mushrooms before making them commercially available. Although not legally required, he plans to seek approval from the Food and Drug Administration (FDA) and Environmental Protection Agency (EPA).

Edited waxy corn may find its way into the food system much sooner than white button mushrooms, if not as human food than as fodder for the growing number of livestock around the world. Meanwhile, these livestock are also undergoing genetic edits as researchers use the same tools to make animals healthier, meatier, and more productive.

Pigs harbor a lot of diseases and there are few as bad as porcine reproductive and respiratory syndrome (PRRS). It causes pregnant mothers to miscarry and makes it difficult for piglets to breathe. Its a problem for the pig farmers as well. Every year, the PRRS virus costs the industry nearly $1.6 billion dollars in Europe and another $664 million in the US.

The impacts of the disease for producers are often devastating, said Jonathan Lightner, Chief Scientific Officer at biotech company Genus. And the impacts on the animals themselves are terrible.

If we could integrate the polled phenotype into the dairy system, that would eliminate dehorning for at least seven or eight million animals a year.

But Lightner and his team are working on a solution. In December 2015, scientists at Genus and the University of Edinburghs Roslin Institute demonstrated how CRISPR could remove the CD163 molecule, a pathway through which the PRRS virus infects pig. Just last month, the researchers refined their work to remove just the portion of the gene that directly interacts with the virus. Lab tests, as published in a paper in the journal PLOS Pathogens, have shown that DNA in cells removed from these pigs successfully resist the virus. Next steps in the study will test whether the pigs themselves are resistant to the virus.

Swine are also the subject of research at Seoul National University in South Korea, where scientists led by Jin-Soo Kin are using a different gene-editing tool called TALEN to create meatier, double muscle pigs by removing a gene that inhibits muscle growth. We could do this through breeding, Kin told Nature back in 2015, but then it would take decades.

In fact, farmers have developed similar traits through breeding Belgian Blues, a type super-sculpted beef cattle prized for its lean meat and beefy build. It took over a hundred years to establish those traits in the breed.

Researchers at University of California, Davis and a startup called Recombinetics are using the same TALEN gene editing technique to cut decades down to days, removing the horned gene from common dairy cows and inserting the one that makes Angus beef cattle naturally dehorned or polled. Polled cattle are desirable because they pose less threat to their handlers and to each other. But, as Tad Sonstegard, Chief Science Officer of Acceligen (a Recombinetics subsidiary) explained, polled cattle in certain breeds are simply less productive.

Gene editing ala CRISPR/Cas9 has allowed scientists to not only produce polled (hornless) cows, but also cows that are immune to common diseases, such as tuberculosis. (Photo: Gregory Urquiaga/UC Davis)

The issue is that the top [dairy] bulls that everyone wants are horned, Sonstegard said. The animals that are polled that already exist have a difference of about $250 over their lifetime. If youre running a thousand head dairy [operation], thats a lot of money.

What many ranchers do instead is dehorn their cattle, a stressful practice when anesthesia is used, a painful practice when it isnt, and a significant expense for the ranchers either way.

If we could integrate the polled phenotype into the dairy system, that would eliminate dehorning for at least seven or eight million animals a year, Sonstegard said. If you include beef, thats up to fifteen million.

Recombinetics has already bred a couple gene-edited calves, which are undergoing care and monitoring at UC Davis. But, before any gene-edited cows produce the milk in our grocery stores, Sonstegard said scientists would need to prove that milk from these cows is similar to horned and polled cows that havent been gene edited. That would be simple though, he said, it would turn out the same.

As the global population grows, so does the demand for food. Meanwhile, farmers around the world face food scarcity generated in part by a changing climate that makes caring for plants and livestock an increasingly difficult task.

But CRISPR-like tools may be able to help.

On the plant side were looking at ways to breed plants that are more drought tolerant or in other ways can better survive the stresses of climate change, Haurwitz said. I think thats incredibly valuable and important as we look at the exploding global population. Caribou has also partnered with Genus in its project to breed PRRS virus resistant pigs.

Beyond his work at Recombinetics, Sonstegard sits on the scientific advisory board of the Centre for Tropical Livestock Genetics and Health, a Gates Foundation-backed initiative to improve the genetics of native livestock in tropical regions. Most productive livestock breeds cant survive the heat or diseases present in tropical environments, and breeds native to tropical environments havent had the same selective breeding programs that generate highly productive livestock.

Will CRISPR be used primarily for patenting foods in ways that fit in existing corporate profit models?

Most of the indigenous animals have not been under strict artificial selection, Sonstegard said. Its all been done anecdotally, since most farmers dont have that many cows and their systems arent that big. Meanwhile, most of the new DNA introduced to these herds is left over semen from bulls in developed countries, according to Sonstegard. Its cheap, he said, and no one in the developed country wants it anymore, so they ship it overseas.

There are a couple possible approaches to strengthening these indigenous breeds. One way would be to edit the DNA of bulls from productive breeds so that theyre more temperature tolerant and disease resistant within tropical climates. Those bulls could then be introduced to the native herds to reproduce and spread their productive genes. Alternatively, the DNA of indigenous bulls could be edited with genes likely to improve productivity of the herd, including milk production and carcass yield.

Right now the trend in those countries is that theres a linear growth in livestock numbers, Sonstegard said, because theyre not improving production but demand is increasing, so they just make more animals.Thats not sustainable.

Researchers are also using CRISPR to save dying and endangered species. This month some of Sonstegards colleagues published a paper showing they could develop surrogate hens that could help raise endangered species of birds. And in Florida, where an invasive disease known as citrus greening is decimating the states iconic orange industry, University of Florida scientists are using CRISPR to develop varieties of orange trees immune to the disease, according to the Tampa Bay Times.

But not everybody is so gung-ho.

UC Davis geneticist Alison Van Eenennaam, who collaborates with Recombinetics on gene-editing polled cows, is absolutely optimistic about the tool I think it can be used for very useful things, she said. Rather than ask why we should use, lets ask how. but shes also careful not to overstate the potential of gene editing. When asked whether the technology could be used to address world hunger, she said, I kind of think that idea is polyamorous. Show me anything that can magically solve world hunger. Lets not oversell this technology. Its useful but its useful for a fairly discreet purpose at this stage, which is making edits to a [gene] sequence that we know has a particular effect.

And CRISPR, of course, has its skeptics. Stacy Malkan, Co-Director of U.S. Right to Know, a nonprofit that calls for transparency and accountability in the food system, is both concerned about the inherent risk involved in gene editing and suspects it could ultimately perpetuate an already imbalanced food system.

Theres really no big difference between [gene editing] and conventional breeding.

Will CRISPR be used primarily for the purpose of patenting foods in ways that fit in existing corporate profit models, she asked, for example, to engineer commodity crops to withstand herbicides, or to engineer livestock to fit better in unhealthy confined feeding operations? Or will it be used to engineer foods that have consumer benefits? Will there be labeling, and safety assessments? There are many questions. Right now we hear a lot of marketing hype about possible benefits of CRISPR, but we heard the same promises about first-generation GMOs for decades and most of those benefits have not panned out.

For scientists like Van Eenennaam, the GMO discussion is over. Frankly, she said, Im over the debate. If someone isnt convinced by the evidence that every single major scientific society in the world says its safe, than nothing Im going to say is going to convince them any differently. When it comes to gene-edited organisms, most scientists are even more insistent about its safety. Theres really no big difference between [gene editing] and conventional breeding, Van Eenennaam added.

But there isnt complete consensus. Malkan points to an interview she recently had with Michael Hansen, senior scientist from Consumers Union, in which Hansen said of CRISPR-like gene editing tools, These methods are more precise than the old methods, but there can still be off-target and unintended effects. When you alter the genetics of living things they dont always behave as you expect. This is why its crucial to thoroughly study health and environmental impacts, but these studies arent required.

From Sonstegards perspective, mutations and off-target effects occur naturally anyway, and gene editing simply offers a more precise approach than selective breeding.

Still, Malkan and others have their reservations, grounded in the idea that its too early to determine the side effects. CRISPR is a powerful research tool for helping scientists understand genetics, how cells react, how entire plants and systems react, she said. In my view these experimental technologies should be kept in the lab, not unleashed in our food system, until those systems are better understood.

See more here:
CRISPR Gene Editing and the DNA of Future Food | Digital Trends

CRISPR/Cas9 is a gene editing technology thats revolutionizing science at a breathtaking pace.

One of its most exciting, taboo, and controversial applications is tweaking the genes of eggs, sperm, or early embryos to alter a human life. This could one day mean the ability to create smarter or more athletic humans (yes, designer babies), but also the chance to knock out disease-causing genetic mutations that parents pass on to their children. Were talking about eliminating mutations linked to diseases like breast and ovarian cancers or cystic fibrosis.

On Wednesday, a team of scientists reported that they have made major progress toward proving the latter is possible.

In a paper published in the prestigious journal Nature, a team led by Shoukhrat Mitalipov of Oregon Health and Science University described how it used CRISPR/Cas9 to correct a genetic mutation thats linked to a heart disorder called hypertrophic cardiomyopathy in human embryos. And they did it without the errors that have plagued previous attempts to edit human embryos with CRISPR.

To be clear, the new work from OHSU was an experiment the point was to test a concept, and the embryos used were never implanted into a womans uterus.

But the researchers were ultimately able to show that CRISPR/Cas9 can do what they hoped it would do. It cut the mutant gene sequence, prompted the embryos to repair the DNA with healthy copies of the gene, and eliminated the disease-causing mutation altogether from many of the embryos.

Lets pause for a minute and make sure were clear on what CRISPR/Cas9 is. You can read our full explainer here, but in a nutshell, its essentially a clever system built into bacterial DNA that allows them to recognize and fend off attackers, usually viruses. The way it works, as Brad Plumer described it, is that special enzymes in the CRISPR sequences known as Cas9 carry around stored bits of viral genetic code like a mug shot. When they find a match to the code, they will chop up the DNA and neutralize the threat.

The real breakthrough, which appeared in a series of landmark papers published in 2012 and 2013, was figuring out that it was possible to program CRISPR/Cas9 to find any kind of DNA code, not just viruses, and get the enzymes to snip it.

Mitalipov and colleagues created embryos in the lab with sperm from a carrier of the disease-causing mutation in the MYBPC3 gene, and eggs from 12 healthy donors. And they sent CRISPR/Cas9 into the fertilized egg.

As the embryos developed, they found that after CRISPR/Cas9 cut the sequence in the embryo DNA with the problematic gene. In most cases the embryos repaired the breaks with a healthy copy of the gene from the maternal donor.

In all, 36 out of 54 embryos ended up with mutation-free copies of MYBPC3. (Another, slight different round of the experiment yielded 42 out of 58 embryos with mutation-free copies of the gene.) Which means that had those embryos become children, the children would have had practically no chance of developing hypertrophic cardiomyopathy. Thats pretty significant since this a disease that affects one in 500 people and can cause sudden cardiac death and heart failure. If one parent has a mutant copy of MYBPC3, their child has a 50 percent chance of inheriting the condition.

These results are also promising for people (mainly older women and couples) who have a limited number of viable embryos to use to get pregnant with in vitro fertilization. Currently, reproductive medicine doctors use something called preimplantation genetic diagnosis, or PGD, to identify embryos with harmful mutations. And when they find embryos with mutations linked to disease, they often discard them, which can leave patients with few healthy embryos to try to transfer into the womb. (Transfer success rates are overall pretty low.)

The researchers say that in the future, their technique could be used with PGD to help fix the mutations in embryos that otherwise would be discarded, giving women and couples more embryos to transfer and a better chance of getting pregnant.

Were not ready for gene editing in embryos that would be implanted for pregnancy anytime soon. But this is a big advance because the researchers got stronger results than anyone who has ever tried to target disease-causing genes with CRISPR-Cas9 before.

And while the experiment focused only on this particular gene and disease, the researchers say they feel confident the technique would work for many of the thousands of other inherited disorders out there linked to one mutation because their approach has so far proved to be efficient, accurate, and safe.

But in a press conference on Tuesday, one of the co-authors, Paula Amato, an OB-GYN doctor at OHSU, stressed that many more safety tests would be needed before proceeding with a clinical trial. We want to replicate the study with other mutations and other [sperm and egg] donors, she said. In particular, she said shes interested in seeing if it works on BRCA1 and 2, mutations that increase the risk of breast and ovarian cancers.

Other researchers, including Nerges Winblad and Fredrik Lanner at Karolinska Institutet in Sweden, who wrote an accompanying article in Nature, are encouraged by the results but still cautious about the safety of the technology. They zeroed in on issues that have shown up in previous studies: off-target effects, or undesirable mutations in genome regions close to the targeted sequence, and mosaicism, where not all embryo cells make the desired changes. According to Winblad and Lanner, researchers will have to keep showing that they can reliably avoid these and other abnormalities in edited embryos before [the technology] can be used as a therapy for inherited diseases.

Amato and her co-authors said theres also plenty of room for other improvement. Some of their embryos DNA ended up with unintended additions or deletions. So their goal would be to get 80 to 90 percent of a large group of embryos mutation-free to ensure that the technique is reliable before attempting a clinical trial.

Again, this wasnt the first time scientists had tried to use CRISPR to edit human embryos. Chinese researchers have done it twice: once in 2015 to modify a gene linked to the blood disorder called beta thalassaemia, and then in 2016 to make genes resistant to HIV. But both of these experiments were smaller, and one used abnormal embryos while the other used immature eggs. And the results from both were messy, suggesting that embryo editing had a long, hard road ahead.

It was precisely those messy results, along with a host of other concerns, that prompted the Organizing Committee for the International Summit on Human Gene Editing at the National Academies of Sciences, Engineering, and Medicine to advise researchers in December 2015 to be extremely cautious about editing sperm, eggs, and embryos (known collectively as the human germline). Then in a report in February, it said clinical trials on human genome editing might one day be allowed, but in the meantime, researchers could attempt to correct mutations that cause a serious disease or condition and only when no reasonable alternatives exist. And definitely no research on enhancement of human traits like intelligence or strength for now.

At present, the US government does not fund any genomic editing of human embryos. (Mitalipov and his colleagues got funding from their university this new study.) And the Food and Drug Administration is prohibited by Congress from considering any clinical trials related to genetic editing of eggs, sperm, or embryos.

The impressive new findings in Nature raise huge questions about how the US should proceed with this field of research. How soon to allow clinical trials, for instance?

I believe [the National Academies] can reconsider what mutations and what cases the gene corrections can be used and must be used to allow clinical trials in the future to go forward, said Mitalipov. We may not be in agreement with the committees. The work is back and forth, and the committees hopefully will consider new options.

He added that hed be willing to move this research to the UK, if necessary. He also is sensitive about how his results are portrayed, given the American publics reticence, and in some cases fear, about genetic modification.

I dont like the word editing, he said. We didnt edit or modify anything. ... We used CRISPR to correct, using existing maternal genes.

Other CRISPR researchers have weighed in about where the field should go from here.

In my opinion, we still need to respect the recommendations in the [National Academy of Sciences] report published in February that recommended refraining from clinical use of human germline editing until and unless theres broad societal consensus about the value, Jennifer Doudna, a UC Berkeley molecular biologist and a leading CRISPR researcher, told the Los Angeles Times.

It may be quite a while before a clinical trial is approved. In the meantime, any prospective parents who want to avoid passing on disease-causing genes to their kids will have to continue to use PGD during in vitro fertilization.

As weve reported, scientists from myriad fields are using CRISPR to try to grow better food, destroy viruses, and clean up the environment.

Hank Greely explains for Vox why in 20 to 40 years, most Americans wont have sex to reproduce.

See the original post here:
Scientists successfully used CRISPR to fix a mutation that ...

Sharifnia, T., et. al. (2017) Cell Chemical Biology. 24:1075-1091. https://www.ncbi.nlm.nih.gov/pubmed/28938087

Rare cancers have traditionally been difficult to study due to low incidence and limited sample availability. However, new technologies, such as sequencing, have allowed for a greater understanding of the underlying genetic causes. In tandem with sequencing technologies, CRISPR/Cas and small molecule screens have allowed researchers to rapidly screen rare cancers for possible mechanisms and treatments.

Sharon Begley, STAT, 25 September 2017, https://www.statnews.com/2017/09/25/nobel-prize-predictions/

The season of Nobel Prize awards has arrived, and with it comes a slew of predictions. This year, STAT has identified who they believe has the best chance of winning the Nobel Prize in Medicine; including the CRISPR crowd of Emmanuelle Charpentier, George Church, Jennifer Doudna, and Feng Zhang. The only problem being each Nobel Prize can only be awarded to three people.

Rachael Lallensack, Nature News, 18 September 2017, http://www.nature.com/news/crispr-reveals-genetic-master-switches-behind-butterfly-wing-patterns-1.22628

Two new studies in the Proceedings of the National Academy of Sciences (http://www.pnas.org/content/early/2017/08/29/1709058114, http://www.pnas.org/content/early/2017/08/29/1708149114) provide insight into butterfly wing color. The studies identified two genes, WntA is responsible for creation of the coloring pattern and borders, while optix fills the color within the borders. Understanding butterfly coloration could provide insights into adaptations such as mimicry.

Vella, M.R. et. al. (2017) Scientific Reports 7:11038. https://www.ncbi.nlm.nih.gov/pubmed/28887462

CRISPR/Cas gene drives could be used to eliminate vector-borne diseases such as malaria and Lyme disease. However, release of modified organisms is controversial in part due to unforeseen consequences. Developing strategies for gene drive reversal could prove useful if such problems arise. This paper develops models to evaluate the effectiveness of gene drive counter-measures in order to evaluate their potential use.

Bikard, D., Barrangou, R., (2017) Current Opinion in Microbiology, 37:155-160. https://www.ncbi.nlm.nih.gov/pubmed/28888103

Self-targeting bacteria with CRISPR usually proves fatal. This observation could lead to a new type of antimicrobial where the CRISPR/Cas system is introduced to fight infection. This review discusses how CRISPR/Cas could target bacterial infections, as well as how the system may be delivered to the infection site.

David Nield, Science Alert, 9 September 2017, https://www.sciencealert.com/now-scientists-are-using-crispr-to-change-the-colour-of-flowers

The Japanese morning glory plant has traditionally had violet flowers, however using CRISPR to disrupt a single gene, scientists have altered the flower color to white. White morning glories can be found; however, it took 850 years for the white version to appear. CRISPR accomplished the task in less than 12 months. This is the first time CRISPR has been used to alter flower color in higher plants.

Liu, X., et. al. (2017) Cell 170:1028-1043. https://www.ncbi.nlm.nih.gov/pubmed/28841410

Many genes are regulated by cis-regulatory elements, though the molecular composition of these elements remains unknown. In a new study published in Cell, Liu et. al. describe a new technique called CAPTURE (CRISPR affinity purification in situ of regulatory elements) that uses a biotin labeled dCas9 to isolate cis regulatory elements in an unbiased fashion, allowing for insights into genome structure and function.

Stanford Medicine News Center, 29 August 2017, http://med.stanford.edu/news/all-news/2017/08/online-game-challenges-players-to-design-on-off-switch-for-crispr.html

Researchers at Stanford University School of Medicine have created a new online computer game called Eterna where players design RNA molecules that could act as an on/off switch for Cas9. Molecular biologists at Stanford will then create the most promising molecules and test them in living cells. Researchers aim to have 100,000 players contribute 10 solutions each. As the research team tests the molecules in the lab, they will provide information to the players for further refinement.

Julia Franz and Katie Hiler, WUNC Science Friday, 27 August 2017, http://wunc.org/post/new-developments-human-gene-editing-face-ethical-and-regulatory-quagmire-us#stream/0

Despite the results of Augusts CRISPR edited embryo paper being called into question, its publication has resulted in an increase in the ethics debate. Scientists agree that CRISPR gene editing will continue to improve and society must grapple with the ethical problems. Ira Flatow sits down with the author of the August Nature article and with Kelly Ormond, genetics professor at Stanford University and member of the Stanford Center for Biomedical Ethics, to discuss the results and how to proceed.

Dieter et. al. (2017) BioRxiv, 181255. http://www.biorxiv.org/content/early/2017/08/28/181255

On 02 August 2017, a Nature article claimed a major breakthrough in CRISPR genome editing. Researchers from around the world, including the United States, announced that they had successfully corrected viable human embryos heterozygous for the MYBPC3 mutation that results in heart disease, without mosaicism. Recently, the results of this article have been called into question with the publishing of a BioRxiv article. The authors of the new paper identify other possible mechanisms that could have caused the observed results and suggest additional experiments to effectively prove CRISPR gene editing.

Read the rest here:
CRISPR Update CRISPR Updates, News and Articles

Gene-editing technology CRISPR has revealed a clue in how human embryos begin to develop, possibly reducing the risk of miscarriage in those crucial first few weeks of pregnancy.

CRISPR Cas9 can modify or snip out genetic defects thought to contribute to miscarriage, but until now it wasnt clear why some embryos continued to form into a fetus and others did not. However,findings, published Wednesday in the journal Nature, hold genetic clues.

British scientists conducting the study found that a certain human genetic marker called OTC4 played an important role in the formation and development in the early stages of embryonic development. The scientists used CRISPR Cas9 to knock out this important gene in days-old human embryos and found that without it, these embryos ceased to attach or grow properly.

The findings could not only help us better understand why some women suffer more miscarriages than others, but it could also potentially greatly increase the rate of successful in vitro fertilization (IVF) procedures.

IVF is sometimes the only way a couple can make a baby using their own genes, but even with technological improvements over the years, the rates of success are still poor.Only about 36 percent of IVF cycles result in a viable pregnancy, and a mere 24 percent produce a baby, according to the Centers for Disease Control.

Of course, this is not the first time scientists have tested on human embryos. The practice has sparked a fierce international debate, but earlier this year, U.S. scientists used CRISPR technology to cut out a gene known to cause heart defects in three-day old human embryos.

None of the embryos in that study or this latest one were meant to go on to become human beings and were discarded after the study was finished. However, both studies hint at the potential CRISPR could have in the formation of human life in the future.

It will likely take years before putting this breakthrough into practice on viable embryos meant to develop beyond a few days, and theres likely still much more research needed, but it does give hope for those whove suffered a miscarriage and wanting to ensure they can one day carry a healthy baby to full term.

Read more:
CRISPR breakthrough could drop miscarriage rates | TechCrunch

Usually, this type of study is conducted on mice, which are easier to come by and carry less ethical considerations. But, in this case, scientists knocked out the gene in 41 human embryos donated by couples who had undergone in-vitro fertilization (IVF). The researchers claim the switch allowed them to highlight key differences between the role of OCT4 in human and mouse models. The team are hoping their findings can help scientists better grasp why some women suffer more miscarriages than others. Additionally, the study could also increase the rate of successful IVF procedures.

This isn't the first time scientists have used human embryos. Earlier this year, a team of researchers from Oregon became the first to use CRISPR tech to cut out genes that cause inherited diseases in humans. Before that, scientists in China utilized the technique to repair a gene that can bring about a fatal blood disorder.

The new study is being hailed as a compelling first step. "We were surprised to see just how crucial this gene is for human embryo development, but we need to continue our work to confirm its role," Norah Fogarty of the Francis Crick Institute told CNN. "Other research methods, including studies in mice, suggested a later and more focused role for OCT4, so our results highlight the need for human embryo research."

Read the rest here:
CRISPR gene-editing could result in more successful birth rates

Have you heard? A revolution has seized the scientific community. Within only a few years, research labs worldwide have adopted a new technology that facilitates making specific changes in the DNA of humans, other animals, and plants. Compared to previous techniques for modifying DNA, this new approach is much faster and easier. This technology is referred to as CRISPR, and it has changed not only the way basic research is conducted, but also the way we can now think about treating diseases [1,2].

CRISPR is an acronym for Clustered Regularly Interspaced Short Palindromic Repeat. This name refers to the unique organization of short, partially palindromic repeated DNA sequences found in the genomes of bacteria and other microorganisms. While seemingly innocuous, CRISPR sequences are a crucial component of the immune systems [3] of these simple life forms. The immune system is responsible for protecting an organisms health and well-being. Just like us, bacterial cells can be invaded by viruses, which are small, infectious agents. If a viral infection threatens a bacterial cell, the CRISPR immune system can thwart the attack by destroying the genome of the invading virus [4]. The genome of the virus includes genetic material that is necessary for the virus to continue replicating. Thus, by destroying the viral genome, the CRISPR immune system protects bacteria from ongoing viral infection.

Figure 1 ~ The steps of CRISPR-mediated immunity. CRISPRs are regions in the bacterial genome that help defend against invading viruses. These regions are composed of short DNA repeats (black diamonds) and spacers (colored boxes). When a previously unseen virus infects a bacterium, a new spacer derived from the virus is incorporated amongst existing spacers. The CRISPR sequence is transcribed and processed to generate short CRISPR RNA molecules. The CRISPR RNA associates with and guides bacterial molecular machinery to a matching target sequence in the invading virus. The molecular machinery cuts up and destroys the invading viral genome. Figure adapted from Molecular Cell 54, April 24, 2014 [5].

Interspersed between the short DNA repeats of bacterial CRISPRs are similarly short variable sequences called spacers (FIGURE 1). These spacers are derived from DNA of viruses that have previously attacked the host bacterium [3]. Hence, spacers serve as a genetic memory of previous infections. If another infection by the same virus should occur, the CRISPR defense system will cut up any viral DNA sequence matching the spacer sequence and thus protect the bacterium from viral attack. If a previously unseen virus attacks, a new spacer is made and added to the chain of spacers and repeats.

The CRISPR immune system works to protect bacteria from repeated viral attack via three basic steps [5]:

Step 1) Adaptation DNA from an invading virus is processed into short segments that are inserted into the CRISPR sequence as new spacers.

Step 2) Production of CRISPR RNA CRISPR repeats and spacers in the bacterial DNA undergo transcription, the process of copying DNA into RNA (ribonucleic acid). Unlike the double-chain helix structure of DNA, the resulting RNA is a single-chain molecule. This RNA chain is cut into short pieces called CRISPR RNAs.

Step 3) Targeting CRISPR RNAs guide bacterial molecular machinery to destroy the viral material. Because CRISPR RNA sequences are copied from the viral DNA sequences acquired during adaptation, they are exact matches to the viral genome and thus serve as excellent guides.

The specificity of CRISPR-based immunity in recognizing and destroying invading viruses is not just useful for bacteria. Creative applications of this primitive yet elegant defense system have emerged in disciplines as diverse as industry, basic research, and medicine.

In Industry

The inherent functions of the CRISPR system are advantageous for industrial processes that utilize bacterial cultures. CRISPR-based immunity can be employed to make these cultures more resistant to viral attack, which would otherwise impede productivity. In fact, the original discovery of CRISPR immunity came from researchers at Danisco, a company in the food production industry [2,3]. Danisco scientists were studying a bacterium called Streptococcus thermophilus, which is used to make yogurts and cheeses. Certain viruses can infect this bacterium and damage the quality or quantity of the food. It was discovered that CRISPR sequences equipped S. thermophilus with immunity against such viral attack. Expanding beyond S. thermophilus to other useful bacteria, manufacturers can apply the same principles to improve culture sustainability and lifespan.

In the Lab

Beyond applications encompassing bacterial immune defenses, scientists have learned how to harness CRISPR technology in the lab [6] to make precise changes in the genes of organisms as diverse as fruit flies, fish, mice, plants and even human cells. Genes are defined by their specific sequences, which provide instructions on how to build and maintain an organisms cells. A change in the sequence of even one gene can significantly affect the biology of the cell and in turn may affect the health of an organism. CRISPR techniques allow scientists to modify specific genes while sparing all others, thus clarifying the association between a given gene and its consequence to the organism.

Rather than relying on bacteria to generate CRISPR RNAs, scientists first design and synthesize short RNA molecules that match a specific DNA sequencefor example, in a human cell. Then, like in the targeting step of the bacterial system, this guide RNA shuttles molecular machinery to the intended DNA target. Once localized to the DNA region of interest, the molecular machinery can silence a gene or even change the sequence of a gene (Figure 2)! This type of gene editing can be likened to editing a sentence with a word processor to delete words or correct spelling mistakes. One important application of such technology is to facilitate making animal models with precise genetic changes to study the progress and treatment of human diseases.

Figure 2 ~ Gene silencing and editing with CRISPR. Guide RNA designed to match the DNA region of interest directs molecular machinery to cut both strands of the targeted DNA. During gene silencing, the cell attempts to repair the broken DNA, but often does so with errors that disrupt the geneeffectively silencing it. For gene editing, a repair template with a specified change in sequence is added to the cell and incorporated into the DNA during the repair process. The targeted DNA is now altered to carry this new sequence.

In Medicine

With early successes in the lab, many are looking toward medical applications of CRISPR technology. One application is for the treatment of genetic diseases. The first evidence that CRISPR can be used to correct a mutant gene and reverse disease symptoms in a living animal was published earlier this year [7]. By replacing the mutant form of a gene with its correct sequence in adult mice, researchers demonstrated a cure for a rare liver disorder that could be achieved with a single treatment. In addition to treating heritable diseases, CRISPR can be used in the realm of infectious diseases, possibly providing a way to make more specific antibiotics that target only disease-causing bacterial strains while sparing beneficial bacteria [8]. A recent SITN Waves article discusses how this technique was also used to make white blood cells resistant to HIV infection [9].

Of course, any new technology takes some time to understand and perfect. It will be important to verify that a particular guide RNA is specific for its target gene, so that the CRISPR system does not mistakenly attack other genes. It will also be important to find a way to deliver CRISPR therapies into the body before they can become widely used in medicine. Although a lot remains to be discovered, there is no doubt that CRISPR has become a valuable tool in research. In fact, there is enough excitement in the field to warrant the launch of several Biotech start-ups that hope to use CRISPR-inspired technology to treat human diseases [8].

Ekaterina Pak is a Ph.D. student in the Biological and Biomedical Sciences program at Harvard Medical School.

1. Palca, J. A CRISPR way to fix faulty genes. (26 June 2014) NPR < http://www.npr.org/blogs/health/2014/06/26/325213397/a-crispr-way-to-fix-faulty-genes> [29 June 2014]

2. Pennisi, E. The CRISPR Craze. (2013) Science, 341 (6148): 833-836.

3. Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero, D.A., and Horvath, P. (2007). CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 17091712.

4. Brouns, S.J., Jore, M.M., Lundgren, M., Westra, E.R., Slijkhuis, R.J., Snijders, A.P., Dickman, M.J., Makarova, K.S., Koonin, E.V., and van der Oost, J. (2008). Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960964.

5. Barrangou, R. and Marraffini, L. CRISPR-Cas Systems: Prokaryotes Upgrade to Adaptive Immunity (2014). Molecular Cell 54, 234-244.

6. Jinkek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. (2012) 337(6096):816-21.

7. CRISPR reverses disease symptoms in living animals for first time. (31 March 2014). Genetic Engineering and Biotechnology News. <http://www.genengnews.com/gen-news-highlights/crispr-reverses-disease-symptoms-in-living-animals-for-first-time/81249682/> [27 July 2014]

8. Pollack, A. A powerful new way to edit DNA. (3 March 2014). NYTimes < http://www.nytimes.com/2014/03/04/health/a-powerful-new-way-to-edit-dna.html?_r=0> [16 July 2014]

9. Gene editing technique allows for HIV resistance? <http://sitn.hms.harvard.edu/flash/waves/2014/gene-editing-technique-allows-for-hiv-resistance/> [13 June 2014]

Read the rest here:
CRISPR: A game-changing genetic engineering technique ...

Why it matters to you

Pigs could be a solution to the shortage of transplant organs. CRISPR gene editing makes them safer candidates.

There is a massive shortage of transplant organs worldwide, and scientists are desperate to come up with a solution whether that be boosting patients immune systems to let them accept otherwise incompatible organs, or creating technology for preserving organs after they are harvested. A new international research initiative has another approach: Using CRISPR gene editing on pigs to make them into safe organ donor candidates for humans.

The reason pigs are desirable as possible sources of organs is that their organs are similar to humans in both size and anatomy. Unfortunately, they also carry viruses known as porcine endogenous retroviruses (PERVs) embedded in their DNA. As this research demonstrated, this can be passed on to humans, although gene editing can be used to eradicate it.

Currently, the major problem of human transplants is the great shortage of transplantable human organs, Lin Lin, a researcher in the department of biomedicine at Denmarks Aarhus University, told Digital Trends. While using pig organs, we can in principle use as many as we need. Eradicating PERVs makes porcine organs safer for human transplants. However, there are still several other barriers that we have to cross in order to make pig organs better for human transplants. This is now achievable with the great development in CRISPR gene editing.

Using an optimized CRISPR-Cas9 gene editing technology and porcine somatic cell nuclear transfer, this work successfully generated viable pigs that are 100 percent PERV-inactivated.Thirty-seven PERV-inactive piglets have so far been born, with 15 remaining alive. The oldest of these is four months old, which means it will need to be monitored for a longer period of time to make sure it suffers no ill-effects.

The next major step is to solve the problem of vigorous immune responses, such as complement activation, coagulation and thrombosis, triggered by xenotransplantation, Lin said. Many previous works have demonstrated that the immunological incapability can be alleviated through tailoring the pig genome. Thus, a serial of very sophisticated gene editing and modifications will be further introduced into the PERV-inactivated pigs and tested in higher primates.

A paper describing the research was recently published in the journal Science.

See more here:
Thanks to CRISPR, gene-edited pigs could be organ donors for ...

B

ioengineer Luhan Yang swiped through the photos on her phone until she got to one that made her beam: It showed her crouching down by a pudgy, wide-eyed newborn she calls my baby.

This newborn is a pig, and its the first to be born with dozens of genetic changes that could enable scientists to turn swine into a source of organs for human transplants, Yang and her colleaguesreported on Thursday in Science.

Theynamed the piglet Laika, after the first dog to orbit Earth in 1957. The new Laika, born this year in China after numerous miscarriages and other setbacks, could be a pioneer in her own right. Using the genome-editing technology CRISPR-Cas9, Yang and her team at the biotech startup eGenesis knocked out pig DNA that has long been considered a deal-breaker for efforts to use pigs as organ donors. Laika and 36 other designer piglets are completely free of it.

advertisement

There are additional Olympic-level hurdles to overcome before people facing death from organ failure get replacement kidneys, hearts, livers, or lungs from the species that provides their bacon and pork chops.Other genetic changes will be necessary. And regulators require stringent tests in lab primates before a single patient could get a CRISPRd pig organ; that will take years.

But after decades of dashed hopes, experts say, xenotransplantation might actually be in the offing.

Its an elegant tour de force of genetic engineering, so my hat is off to them, said Dr. A. Joseph Tector, of the University of Alabama, Birmingham, who has also made genetically modified pigs aimed at producing transplantable organs. But if you want to move xenotransplantation to the hospital, there are many more things youll have to do.

Doctors wont have to do much persuasion, however, to get patients to accept organs from another species. There is so much desperation among people on transplant lists, and 20 a day are dying as they wait, said Dr. Adam Griesemer, a xenotransplantation researcher and transplant surgeon at Columbia University Medical Center. This could be a path to a transplant for them. Colleagues keep asking me when were going to do it.

Pigs are scientists first choice because their organs and physiology are pretty close matches to humans, and they come with less ethical baggage than, say, chimps or baboons. But for years, the path to xenotransplantation has been paved with disappointment. Pig organs with genetic changes, transplanted into baboons and other lab animals,kept failing within weeks, even though the recipients received immune-suppressing drugs to prevent organ rejection.

Yang believes that CRISPR can accomplish what previous approaches have not: make multiple, simultaneous changes in pig DNA so that the animals organs work, and work safely, in people.

The team at Massachusetts-based eGenesis, working with scientists in China, used the Dolly recipe to clone pigs. They started with cells from adult pigs, and used an electrical jolt to fuse them with pig ova whose DNA had been removed. They grew the resulting embryos in lab dishes and then transferred healthy ones to sows, hoping for pregnancies.

The adult cells were not as nature made them, however. In a key step, the scientists used the genome-editor CRISPR to cripple all 25 copies of PERV genes DNA in the pig genome that makes potentially dangerous viruses that could infect anyone who receives a pig organ. (PERV stands for porcine endogenous retroviruses.) Initially, in about one-third of the CRISPRd pig cells, the PERV genes were almost all gone. In most of the rest, CRISPR missed its mark. That wasnt unexpected; for all the hype around CRISPR, it isnt perfect.

The unwelcome surprise was that cells that were effectively CRISPRd the ones the scientists needed to clone designer pigs were dying like orchids in the tundra. Apparently, in its zeal to attack so many PERV genes, CRISPR had shredded the cells genomes fatally.

Its quite a problem, when you move to so many targets, said Yang, the chief scientific officer at eGenesis. If there are multiple cuts in the genome at the same time, chromosomes rearrange themselves. That can happen when you make two or three [CRISPR edits], and were dealing with 25.

The eGenesis scientists, many of them alums of George Churchs lab at Harvard Medical School, scrambled for a solution. They eventually stumbled on a cocktail of molecules that both increased the number of PERV targets that CRISPR hit and, even better, kept the well-CRISPRd cells alive. We were able to get cells to grow even with very aggressive gene editing, Yang said: 100 percent of the cells doused with the chemical cocktail were 100 percent PERV-free.

As is typical with cloning, very few of the cloned embryos were healthy enough to implant into sows, and few implanted embryos resulted in births. Crucially, however, of the 37 piglets born from 17 sows, all were PERV-free. And CRISPR did not change any DNA it wasnt supposed to; there were no off-target effects.

The oldest pigs are nearly 5 months old, or adolescents; 15 remain alive. The rest were killed so the scientists could see whether their organs were developing normally.

So far, so good, Yang said, showing that pigs dont need PERVs to live: Weve shown you can produce PERV-free pigs which could serve as a source for future xenotransplants.

Among eGenesiss next experiments: see if the pigs are fertile and, if so, whether their CRISPRd genetic changes, including inactivating PERVs, are inherited. That could provide an easier source of transplantable organs than cloning.

Other scientists have also used CRISPR to produce pigs with altered genomes, including pigs in which a genethat triggers organ rejection was eliminated. Last year, scientists announcedthat hearts from genetically-modified pigs survived in baboons for up to 945 days, a record.

UABs Tector and his colleagues, with financial backing from United Therapeutics Corp., are using CRISPR not on PERVs but on other pig genes. Knocking out threein particular could protect pig organs from being attacked by the human immune system, he said; lab macaques that received kidneys from the pigs have survived as long as 499 days. We have a pig we are very confident we can make work for kidney transplants, Tector said.

There is disagreement about whether pig organs would have to be PERV-free to be successfully transplanted into people. Tector said transplant patients could take anti-retroviral drugs, just as they take immune-suppressing drugs, to kill the viruses.

Nevertheless, eGenesis scientists achievement with their 25 DNA edits, the eGenesis pigs set the record for genome modifications suggests that however many edits are needed to make pigs into organ donors might be feasible. The challenge is to identify which pig genes are necessary and sufficient to change so that the animals organs have a shot at working in people.

Senior Writer, Science and Discovery

Sharon covers science and discovery.

More:
Birth of CRISPR'd pigs advances hopes for turning pigs into ...

CRISPR or CRISPR Cas9 is commonly used to refer to a revolutionary genome editing technology that enables efficient and precise genomic modifications in a wide variety of organisms and tissues.

Definition: Clustered Regularly Interspaced Short Palindromic Repeat or CRISPR (pronounced 'crisper') was identified in a prokaryotic defence system. CRISPR are sections of genetic code containing short repetitions of base sequences followed by spacer DNA segments

Identified in archaea and bacteria, short nucleic acid sequences are captured from invading pathogens and integrated in the CRISPR loci amidst the repeats. Small RNAs, produced by transcription of these loci, can then guide a set of endonucleases to cleave the genomes of future invading pathogens, thereby disabling their attacks.

Definition: CRISPR ASsociated protein 9 (Cas 9) is an endonuclease used in an RNA-guided gene editing platform. It uses a synthetic guide RNA to introduce a double strand break at a specific location within a strand of DNA

Cas9 was the first of several restriction nucleases (or molecular scissors) discovered that enable CRISPR genome editing. The CRISPR Cas9 mechanism has since been adapted into a powerful tool that puts genome editing into the mainstream.

In the laboratory, CRISPR Cas9 genome editing is achieved by transfecting a cell with the Cas9 protein along with a specially designed guide RNA (gRNA) that directs the cut through hybridization with its matching genomic sequence. When the cell repairs that break, errors can occur to generate a gene knockout or additional genetic modifications can be introduced. Our CRISPR gene editing technology is particularly good for the efficient generation of complete knockout of genes on multiple alleles.

Use of wild-type Cas 9 has been shown to lead to off-target cleavage, but a modified version introduces only single strand nicks to the DNA, which in pairs still stimulate the repair mechanisms while significantly decreasing the risk of off-target cutting.

Horizon has licensed gene editing IP from Harvard University, the Broad Institute and ERS Genomics with the goal of being able to ensure that we will be able to offer uninterrupted use of CRISPR tools to our customers. Our scientists have extensive knowledge of CRISPR technology including the benefits of using each Cas9 structure.

Other Gene Editing Systems

Genome editing can be achieved using the widely used S. Pyogenes (spCas9), and also utilising CRISPR Cas 9 protocol for S. Aureus (scCas9), Cpf1, HiFi Cas9, Nickase Cas9, Nuclease Cas9, NgAgo gDNA and even synthetic spCas9 with alternative PAM sites.

Our genome editing knowledge also includes rAAV and ZFNs.

Continue your CRIPSR/Cas9 research with ourpopular education and training webinars:

Find out more about our exciting upcoming eventwhere the future of CRISPR will be discussed:

The CRISPR Forum 2017

Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P. 2007.CRISPR provides acquired resistance against viruses in prokaryotes. Science 315(5819): 1709-1712.

Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. 2012.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096): 816-821.

Continued here:
CRISPR - CRISPR-Cas9 | Gene Editing