What Is CRISPR? – CB Insights
Posted: September 24, 2018 at 6:43 pm
CRISPR. What is it? And why is the scientific community so fascinated by its potential applications? Starting with its definition, we explain how this technology harnesses an ancient bacteria-based defense system and how it will impact the world around us today.
Imagine a future where parents can create bespoke babies, selecting the height and eye color of their yet unborn children.In fact, all traits can be customized to ones preferences: the size of domestic pets, the longevity of plants, etc.
It soundslike the backdrop of a dystopian science fiction novel. Yet some of this isalready happening.
Since its initial discovery in 2012, scientists have marveled at the applications of CRISPR (also known as Cas9 orCRISPR-Cas9).
And with a Jennifer Lopez-produced bio-terror TV drama called C.R.I.S.P.R. on the horizon, CRISPR has reached a new peak in interest from outside the scientific community.
CRISPR may revolutionize howwe tackle some of the worlds biggest problems, like cancer, food shortages, and organ transplant needs.Recent reports even examineits useasa highly efficient disease diagnostics tool. But, as with any new technology, it may also cause new unintended problems.
Changing DNA the code of life will inevitably come with a host ofimportant consequences. But society and industry cant have this conversation without understanding the basics of CRISPR.
In this explainer, we dive into CRISPR, from a simple explanation of what exactly it is to its applications and limitations.
CRISPR is adefining feature of the bacterial genetic code andits immune system,functioningas a defense system that bacteria use to protect themselves against attacks from viruses. Its also used by organisms in the Archaea kingdom (single-celled microorganisms).
The acronym CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. Essentially, it is a series ofshort repeating DNA sequences withspacers sitting in between them.
In short,bacteria usethese geneticsequences to remember each specific virus that attacks them.
They do this byincorporatingthe virus DNA into their own bacterial genome. Thisviral DNA ends up as the spacers in the CRISPR sequence.This method then gives thebacteria protection or immunity when a specific virus tries to attack again.
Accompanying CRISPR are genes that are always located nearby, called Cas (CRISPR-associated) genes.
Once activated, these genes make special proteins known as enzymes that seem to have co-evolved with CRISPR. The significance of these Cas enzymes is their ability to act as molecular scissors that can cut into DNA.
To recap: in nature,when a virus invades bacteria, its unique DNA is integrated into a CRISPR sequence in the bacterial genome. This means that the next time the virus attacks, the bacteria will remember it and sendRNA and Cas to locate and destroy the virus.
While there are other Cas enzymes derived from bacteria that cut out viruses when they attack bacteria, Cas9 is the best enzyme at doing this in animals. The widely-known term CRISPR-Cas9 refers to a Cas variety beingused to cut animal (and human) DNA.
Inharnessing this technology, researchers have added a new step: after DNA is cut by CRISPR-Cas9, a new DNA sequence carrying a fixed version of a gene can nestle into the new space. Alternatively, the cut can altogether knock out ofa particular unwanted gene for example, a gene that causes diseases.
Oneway to think about CRISPR-Cas9 isto compare it to theFind & Replace function in Word: itfinds thegenetic data (or word)you want to correct and replaces it with new material. Or, as CRISPR pioneer Jennifer Doudna puts it in her book A Crack In Creation: Gene Editing and the Unthinkable Power to Control Evolution, CRISPR is likea Swiss army knife, with different functions depending on how we want to use it.
CRISPR research has moved so fast that its already gone beyond basic DNA editing. In December 2017, the Salk Institute designed a handicapped version of the CRISPR-Cas9 system, capable of turninga targeted gene on or off without editing the genome at all. Going forward, this kind of process could ease the concerns surrounding the permanent nature of gene editing.
These are the 3 key players that help theCRISPR-Cas9 tech do its work:
Below, we illustrate how these parts come together to create a potential therapy.
Please click to enlarge.
The guide RNAserves as the GPS coordinates for finding the piece of DNA you want toedit and zeroes in on the targeted part of the gene. Once located, Cas9, the scissors, makes a double stranded break in the DNA, and the DNAyou want to insert takes its place.
The implications for this are vast.
Yes, this technology will disrupt medical treatment. But beyond that, it could also transform everything from the food we eat to the chemicals we use as fuel, since these may be engineered through gene technology as well.
Feng Zhang, PhD, from the Broad Institute of MIT and Harvard, describedCRISPR using a helpful nursery rhyme. We can imagine a certainDNA sequence that is fixed in this way:
Twinkle Twinkle Big Star Twinkle Twinkle Little Star
In this process:
The CRISPR sequence was first discovered in 1987. But its function would not be discovered until 2012.
Keypeople involved in the initial discovery of the bacterial CRISPR-Cas9 systems function include Jennifer Doudna, PhD at University of California, Berkeley, and French scientist Emmanuelle Charpentier, PhD. Through their strategic collaboration, they ushered in a new era of biotechnology.
Another important figure is Feng Zhang, PhD, who was instrumental in figuring out CRISPRs therapeutic applications using mice and human cells in 2013.Harvard geneticist George Church, PhDalso contributed to early CRISPR research with Zhang.
All four researchers went on to play crucial roles in setting up someof the most well-funded CRISPR therapeutic startups, includingEditas Medicine, CRISPR Therapeutics, and Intellia Therapeutics.All 3 of these companiesIPOed in 2016 and are in the drug discovery/pre-clinical stage of testing their respective CRISPR therapeutic candidates for various human diseases.
Before CRISPR was heralded asthegene editing method, two other gene-editing techniques dominated the field: Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs). Research efforts using these tools are still ongoing.
Like CRISPR, these toolscan each cut DNA. Thought they are generally more difficult to make and use, these tools do offer their own advantages:
Each also has vital therapeutic applications.
Biotech companyCellectis uses the TALEN gene editing technology to make CAR-T therapies for leukemia, whileSangamo BioSciencesmakes ZFNs that can disable a gene known to be key in HIV infection.Notably, each of these companies hold key IP rights to these specific gene-editing methods, which could make it difficult for other biotech companiesto use these tools.
Meanwhile, CRISPR has certainly stolen the spotlight as of late, due to its efficiency, flexibility, and cheap price tag. Itsplausible that CRISPR could face similar IP issues and there are already some IP controversies going on but with such vast applications for this system, research on multiple fronts seems to be moving forward fast.
Every industry can harnessCRISPR as a tool: itcan create new drug therapies for human diseases, help farmers grow pathogen-resistant crops, create new species of plants and animals and maybe even bring back old ones.
Since the initial discovery of CRISPR as a gene-editing mechanism, the list of applications has grown rapidly. Though still in early stages, animal models (i.e. lab animals) have provided key insights into how we may be able to manipulate CRISPR.
Mice have been especially telling when it comes to CRISPRs therapeutic potential. As mammals sharing more than 90% of our human genes, mice have been used as ideal test subjects.
Experiments on mice haveshown that CRISPR can disable a defective gene associated with Duchenne muscular dystrophy (DMD), inhibit the formation of deadly proteins involved in Huntingtons disease, and eliminate HIV infection.
In 2015, Chinese scientists created two super muscular beagles by disabling the myostatin gene, which directs normal muscle development. In the absence of thegene, the beagles displayed muscular hypertrophy, creating dogs which were visibly much more muscular than non-genetically modified ones.
Other CRISPR animal trials haveranged from genetically engineering long-haired goats for higher production of cashmere to breeding hornless cows to avoid the painful process of shearing horns off.
Compared to research involving animals, CRISPR trialsthat edit human DNA have movedmore slowly, largely due to the ethical and regulatory issues at play.
Given the permanent nature of altering a humans genome, the FDA is approaching CRISPR cautiously. Some scientists have even proposed a moratorium on CRISPR trials untilwe have more information on the potentialimpact on humans.
In the US and Europe, 2018 will be the year for CRISPR human trials.
Currently (as of 2/13/18),the University of Pennsylvania is awaiting the FDAs final approval to start a study that would evaluate the safety of using CRISPR for patients with multiple myeloma, melanoma, and sarcoma.
Europe may also see its first human CRISPR study in 2018 withCRISPR Therapeutics study focused on a blood disorder known as beta-thalassemia,which results in abnormal red blood cell production.
While clinical trials involving patient participation are still awaiting regulatory approval, CRISPR has already been applied to both viable and non-viable human embryos.
For example, in August 2017, a team lead by reproductive biologist Shoukhrat Mitalipov of Oregon Health and Science University received private funding to use CRISPR-Cas9 to target a mutation in viable human embryos that causes the thickening of heart muscles. The altered embryos came back 72% mutation-free in the lab (higher than theusual 50% chance of inheritance).
Some critics say the gene editing of embryos is unethical, even if the edited embryos are not destined for transfer and implantation. This type of testing currently does not receive federal funding, but instead relies on private donor funding.
On the other side of the world, Chinese researchersoperate under a different regulatory framework. Some hospital ethics committees can approve studies in as little as one day, with no need to seek approval from a federal agency.
Since 2015, China has been conductinghuman trials using CRISPRto combat various cancers, HIV, and HPV. It is the only country in the world toconduct human trials thus far.
According to ClinicalTrials.Gov, there are 10 active or upcoming CRISPR therapy trials in China, targeting advanced cancers like stage 4 gastric and nasopharyngeal carcinomas. So far results are only anecdotal, and while some participants tumors shrank, no formal results have been made available.
Although possible long-term side effectsarent fully understood,CRISPR is already an option for some patients in China who have exhausted all of the conventional treatments.
Potential high impact industries for CRISPR include medicine, food, agriculture, and the industrial biotech space. BecausetheCRISPR-Cas9 gene-editing system issoeasy to make and use, researchers from a range of scientific disciplines can access it to genetically engineer the organism of their choice.
The future of medicine will be written with CRISPR.
The current drug discovery process is long, given the need to ensure patientsafety and gain a thorough understanding of unintended effects.Moreover, current US regulatory policies often result in a decades-long development process.
However, teamsusing CRISPR can bringcustomized therapies to market more quickly than was previously dreamed, speeding upthe traditional drug discovery process.
Timeline of drug development. Credit: PhRMA
CRISPRscheap price tag and flexibilityallows accurate and fast identification of potential gene targets for efficient pre-clinical testing. Because itcan be used to knock out different genes, CRISPR givesresearchers a faster and more affordableway to study hundreds of thousands of genes to see which ones are affected by a particular disease.
Of course, alongwith providing a more streamlined drug development process, CRISPR offers the possibility of new ways to treat patients.
For example,monogenic diseasesdiseases caused by a mutation ina single gene present an attractive starting point for CRISPR trials. The nature of these illnesses provides an exact target for the treatment: the problematic mutation on a single gene.
Blood-based, single-gene diseases like beta-thalassemia or sickle cell are alsogreat candidates for CRISPR therapy, because of their ability to be treated outside of the body (known as ex-vivo therapy). A patients blood cells can be taken out, treated with the CRISPR system, then put back into the body.
An earlyapplication of CRISPR was pioneered by yogurt company Danisco in the 2000s, when scientists used an early version of CRISPR to combat a key bacterium found inmilk and yogurt cultures (streptococcus thermophilus) that kept getting infected by viruses. At that point, the ins and outs of CRISPRwere still unclear.
Fast forward to today, when climate change will further increasethe need to use CRISPR to protect the food and agriculture industries against new bacteria.For example, cacao is becoming difficult to farm as growing regions get hotter and drier. This environmental change will further exacerbate the damage done by pathogens.
If youve eaten yogurt or cheese, chances are youve eaten CRISPR-ized cells.
Rodolphe Barrangou, former Daniscoscientist & Editor-in-Chief of The CRISPR Journal
To combat this issue, the Innovative Genomics Institute (IGI) at UC Berkeley is applying CRISPR to create disease-resistant cacao. Leading chocolate supplier MARS Inc. is supporting this effort.Gene editing can make farming more efficient. It can curb global food shortages for staple crops like potatoes and tomatoes. And it can create resilient crops, impervious to droughts and other environmental impacts.Regulators have shown little resistanceto gene-edited crops, and the United States Department of Agriculture (USDA) in particular is not regulating the space. This is largely because when CRISPR is applied to crops, theres no foreign DNA being added: CRISPR is simply used to edit a crops own genetics to select for desirable traits.In 2016, the white button mushroom, modified to beresistant to browning, became the first CRISPR-edited organism to bypass USDA. In October 2017, it was announced that agriculture company DuPont Pioneer and the Broad Institute would collaborate for agriculture researchusing their CRISPR-Cas9 intellectual property.
InSeptember 2017, biotech company Yield10 Bioscience got approval for its CRISPR-edited plantCamelina sativa (false flax), which hasenhanced omega-3 oil and is used to make vegetable oil and animal feed.
These are indicationsthat newbreeds of crops could reachmarketsmuch faster than previously thought. Without USDA oversight, these items and other food products could go into production relatively quickly.
This will impact the food we eat, as food items are edited tocarry more nutrients or to last longer on grocery shelves.
Another area currently generating buzz isthe production of leaner livestock.
In October 2017, scientists at the Chinese Academy of Sciences in Beijing used CRISPR to genetically engineer pig meat that had 24% less body fat.
Researchersdid this by inserting a mouse gene into pig cellsin order tobetter regulate body temperature.Although this example technically makes the result a GMO product, it may not be too long before pigs genes are used for the same purpose.
Future versionsof this technology applied to human nutrition will be one area to look out for.
Another key, but less obvious, use of CRISPR lies is in the industrial biotech space. By re-engineering microbes using CRISPR,researcher can create new materials.
How is this relevant to society at large?
From an industrial standpoint, this is big for modifying and creating new chemical products. We can alter microbes to increase diversity, create new bio-based materials, and make more efficient biofuels.From active chemicals in fragrances to those involved in industrial cleaning, CRISPR could have agreat impact here by creating new and more efficientbiological materials.
Jennifer Doudnas first CRISPR startup, Caribou Biosciences, was founded in 2011 for non-therapeutic research purposes across industries. It is one of the key companies providing various industries with the tools to use CRISPR fora range of purposes.
CRISPRs list of potential benefits is a long one. But the technology also brings with it a number of limitations.
Possible unintended effects and all the unknown variables are some of the drawbacks to this newtechnology, while newethicalquestions and controversies are also emerging as human trials near.
When using CRISPRfor human therapies, safety is the biggest issue. As with any new form of technology, researchers are unsure of the entire range of CRISPRs effects.Off-target activity is the main concern here. A single gene editcould cause unintended activity somewhere else in the genome. A possible consequence of this is abnormal growth of tissues, leading to cancer. As more research uncovers new details, this could result in more refined, precise gene targeting.
Another issue is the possibility of mosaic generation.After a CRISPR treatment, a patient could have a mix of both edited and unedited cells a mosaic. As cells continue to divide and replicate, some cells may get repaired, while others wont.
Finally, immune systemcomplications mean that these interventions and therapies may trigger an undesired response froma patients immune system.Early research shows theimmune system may dispose of Cas enzymes before they achieve their purpose, or may have an averse reaction resulting in side effects like inflammation. (In 1999, a patient in the US died of a severe immune reaction, instilling more caution in researchers when it comes to CRISPR trials.)
However, all three of these limitations have some possible solutions.
Different enzymes (molecular scissors) or more precise delivery vehicles can reduce off-target activity. If modified stem cells in egg or sperm (i.e. cells that can become every cell in the human body) are edited, mosaics can be avoided.
With the immune system issue, researchers can isolate different Cas proteins from more obscure bacterial strains that humans dont already have an adaptive immunity to in order to circumvent an unwanted immune response. Meanwhile, ex-vivo therapies, wherescientists take a patients blood cells out of the body and treat them before infusing them back in, can also helpbypass the immune system.
One potential big limitation for CRISPR isthat CRISPR-Cas9 system lacks surgical precision. The Cas enzyme cuts both strands of the DNA double helix, and this double-stranded breakcreates worries over the precision of the cut.
Repairing a defective gene would be like finding a needle in a haystack and then removing that needle without disturbing a single strand of hay in the process.-Jennifer Doudna
While currently the Cas9 enzyme gets the most attentionas the enzyme doing the cutting, scientists are actively pursuing alternatives to find better candidates.
Alternative options include asmaller version of Cas9, or a different enzyme entirely: Cpf1, whichhas become popular due to its easy transport to the targeted DNA location.
Besides using other Cas enzymes, alternate delivery vehiclesfor therapeutic genes are another option. Harmless engineered viruses can carry therapeutic genes to the site of mutation, while lipid nanoparticles can avoid immune system detection, avoiding an immune reaction. Both options present promising avenues of research.
Whentechnology can alter the code of life, its implications are far-reaching as are its controversies. Here we outlinea few of the main controversies surroundingCRISPR.
Originally posted here:
What Is CRISPR? - CB Insights
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