Archive for the ‘Crispr’ Category

Mar. 2015: We are excited to announceGenome Engineering 3.0,a 2-day practical workshop designed to help you use CRISPR-Cas9 system to perform all different types of genome engineering tasks more effectively in your own work.It will be held from May 8-9, 2015 at the Broad Institute (map) in Cambridge, MA, USA.The workshop is open to all and free. For more details, please visit our workshop website, and if you are ready to sign up, you can register here.

Feb. 2014: Collaboration with Osamu Nureki and Hiroshi Nishimasuat University of Tokyoreveals crystal structure of Cas9 in complex with guide RNA and target DNA.

Dec. 2013: New Genome-scale CRISPR Knock-out (GeCKO) functional genomics screening study inShalem* and Sanjana* et al., Science, 2013and companion website.

July 2013: Based on the specificity analysis performed in Hsu et al., Nature Biotechnology 2013, we have released the CRISPR Design Tool. This tool allows users to search for high specificity SpCas9 target sites within DNA sequences of interest.

Apr. 2012: Have a question or need help with trouble shooting? Please visit our newly updated FAQs and Trouble Shooting Tips page.

Feb. 2013: For highest efficiency in genome targeting applications, please use our new chimeric RNA design with longer tracrRNA hybrid.In the earlier paper we useda shorter chimeric RNA design that is not optimal, now the new longer chimeric RNA design is most effective (better than crRNA:tracrRNA pair).This is verified by other published CRISPR paper as well.

Jan. 2013: Please feel free to post your questions to our CRISPR Discussion Forum.

Originally posted here:

CRISPR Genome Engineering Resources | learn, share, and discuss

The CRISPR system

Like zinc fingers and TALEs, CRISPR systems are natural products. However, CRISPR-Cas differs from zinc fingers and TALEs in one crucial aspect that makes it superior for genome editing applications: whereas zinc fingers and TALEs bind to DNA through a direct protein-DNA interaction, requiring the protein to be redesigned for each new target DNA site, CRISPR-Cas achieves target specificity through a small RNA that can easily be swapped for other RNAs targeting new sites.

In nature, CRISPR-Cas systems help bacteria defend against attacking viruses (known as bacteriophage or just phage). They consist of two components, the CRISPR (clustered, regularly interspaced palindromic repeats) array and Cas (CRISPR-associated) proteins. CRISPR sequences bookend short stretches of DNA that bacteria have copied from invading phages, preserving a memory of the viruses that have attacked them in the past. These sequences are then transcribed into short RNAs that guide Cas proteins to matching viral sequences. The Cas proteins destroy the matching viral DNA by cutting it. There are a number of different types of CRISPR-Cas systems in nature, which vary in their components; the CRISPR-Cas9 system uses just a single protein, Cas9, to find and destroy target DNA. In 2015, Zhang and colleagues successfully harnessed a second system, called CRISPR-Cpf1, which has the potential for even simpler and more precise genome engineering.

In early 2011, Feng Zhang was just starting his own research group at the Broad Institute and MIT, where he is an investigator at the McGovern Institute for Brain Research and a faculty member in the departments of Brain and Cognitive Sciences and Biological Engineering.After learning about existing CRISPR researchat a scientific meeting at the Broad, he quickly realized that the system, with a single RNA-guided protein, could be a game changer in genome editing technology. He was already working on DNA targeting methods, having helped to develop the TALE system as a Junior Fellow at Harvard. This system could target and activate genes in mammalian genomes.

Zhang and his team focused on harnessing CRISPR-Cas9 for use in human cells. In January 2013, he reported the first successful demonstration of Cas9-based genome editing in human cells in what has become the most-cited CRISPR paper (Cong et al., Science, 2013). Researchers from George Churchs lab at Harvard University reported similar findings in the same issue of Science (Mali et al., Science, 2013). The Zhang and Church papers showed that Cas9 could be targeted to a specific location in the human genome and cut the DNA there. The cut DNA was then repaired by inserting a new stretch of DNA, supplied by the researchers, essentially achieving find and replace functionality in the human genome.

In September, 2015, Zhang and partners described a different system, Cpf1, which appears to have significant implications for research and therapeutics.The Cpf1 system is simpler in that it requires only a single RNA. The Cpf1 enzyme is also smaller than the standard SpCas9, making it easier to deliver into cells and tissues.

The CRISPR toolbox is continuing to expand rapidly, opening new avenues for biomedical research. Since the first publications in early 2013, the Zhang lab and other researchers have engineered a number of improvements to the system. For example, Cas9 has been modified so that instead of cutting the target DNA, it can turn gene expression on by recruiting transcriptional activators to its genomic location (Konermann, et al., Nature, 2014).

At the Broad Institute, the system has also been used for genome-wide screens to identify genes involved in resistance to cancer drugs and dissect immune regulatory networks. CRISPR has been used to rapidly create mouse models of cancer that arise from multiple gene alterations (Platt et al., Cell, 2014). In 2015, Zhang and his team reported success with Cas9 derived from a different bacterium, Staphylococcus aureus (SaCas9). SaCas9 is smaller than the original Cas9, which has advantages for gene therapy (Ran et al., Nature, 2015).

The Zhang lab has trained thousands of researchers in the use of CRISPR-Cas9 genome editing technology through direct education and by sharing more than 37,000 CRISPR-Cas9 components with academic laboratories around the world to help accelerate global research that will benefit human health. In September 2015, the Zhang lab also began to share Cpf1 components.

Users can obtain guide sequences for knock-outs and transcriptional activation as well as information about genome-wide libraries for CRISPR-based screening. To learn more, visit the Zhang Lab CRISPR Resources at http://www.genome-engineering.org/.

Read this article:

CRISPR | Broad Institute

Q: What is CRISPR?

A: CRISPR (pronounced crisper) stands for Clustered Regularly Interspaced Short Palindromic Repeats, which are the hallmark of a bacterial defense system which forms the basis for the popular CRISPR-Cas9 genome editing technology. In the field of genome engineering, the term CRISPR is often used loosely to refer to the entire CRISPR-Cas9 system, which can be programmed to target specific stretches of genetic code and to edit DNA at precise locations. These tools allow researchers to permanently modify genes in living cells and organisms and, in the future, may make it possible to correct mutations at precise locations in the human genome to treat genetic causes of disease. In September 2015, the Zhang lab demonstrated successful harnessing of a different CRISPR system for genome editing, called CRISPR-Cpf1, which has the potential for even simpler and more precise genome engineering.

Q: Where do CRISPRs come from?

A: CRISPRs were first discovered in archaea (and later in bacteria), by Francisco Mojica, a scientists at the University of Alicante in Spain. He proposed that CRISPRs serve as part of the bacterial immune system, defending against invading viruses. They consist of repeating sequences of genetic code, interrupted by spacer sequences remnants of genetic code from past invaders. The system serves as a genetic memory that helps the cell detect and destroy invaders (called bacteriophage) when they return. Mojicas theory was experimentally demonstrated in 2007 by a team of scientists led by Philippe Horvath.

In January 2013, Feng Zhang at the Broad Institute and MIT published the first method to engineer CRISPR to edit the genome in mouse and human cells.

Q: How does the system work?

A: CRISPR spacer sequences are transcribed into short RNA sequences (CRISPR RNAs or crRNA) capable of guiding the system to matching sequences of DNA. When the target DNA is found, Cas9 one of the enzymes produced by the CRISPR system binds to the DNA and cuts it, shutting the targeted gene off. Using modified versions of Cas9, researchers can activate gene expression instead of cutting the DNA. These techniques allow researchers to study the genes function.

Research also suggests that CRISPR-Cas9 can be used to target and modify typos in the three-billion-letter sequence of the human genome in an effort to treat genetic disease.

Q: How does CRISPR-Cas9 compare to other genome editing tools?

A: CRISPR-Cas9 is proving to be an efficient and customizable alternative to other existing genome editing tools. Since the CRISPR-Cas9 system itself is capable of cutting DNA strands, CRISPRs do not need to be paired with separate cleaving enzymes as other tools do. They can also easily be matched with tailor-made guide RNA (gRNA) sequences designed to lead them to their DNA targets. Tens of thousands of such gRNA sequences have already been created and are available to the research community. CRISPR-Cas9 can also be used to target multiple genes simultaneously, which is another advantage that sets it apart from other gene-editing tools.

CRISPR-Cpf1differs in several important ways from the previously described Cas9, with significant implications for research and therapeutics.

First: In its natural form, the DNA-cutting enzyme Cas9 forms a complex with two small RNAs, both of which are required for the cutting activity. The Cpf1 system is simpler in that it requires only a single RNA. The Cpf1 enzyme is also smaller than the standard SpCas9, making it easier to deliver into cells and tissues.

Second, and perhaps most significantly: Cpf1 cuts DNA in a different manner than Cas9. When the Cas9 complex cuts DNA, it cuts both strands at the same place, leaving blunt ends that often undergo mutations as they are rejoined. With the Cpf1 complex the cuts in the two strands are offset, leaving short overhangs on the exposed ends. This is expected to help with precise insertion, allowing researchers to integrate a piece of DNA more efficiently and accurately.

Third: Cpf1 cuts far away from the recognition site, meaning that even if the targeted gene becomes mutated at the cut site, it can likely still be re-cut, allowing multiple opportunities for correct editing to occur.

Fourth: the Cpf1 system provides new flexibility in choosing target sites. Like Cas9, the Cpf1 complex must first attach to a short sequence known as a PAM, and targets must be chosen that are adjacent to naturally occurring PAM sequences. The Cpf1 complex recognizes very different PAM sequences from those of Cas9. This could be an advantage in targeting some genomes, such as in the malaria parasite as well as in humans.

Here is the original post:

Questions and Answers about CRISPR | Broad Institute

CRISPR or CRISPR Cas 9 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-Cas 9 mechanism has since been adapted into a powerful tool that puts genome editing into the mainstream.

In the laboratory, 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 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 technology 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 research with our CRISPR/Cas9 videos:

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.

Read the original:

CRISPR Gene Editing - CRISPR/Cas9 - Horizon Discovery

On Friday, a team of Chinese scientists used the cutting-edge gene-editing technique CRISPR-Cas9 on humans for the second time in history, injecting a cancer patient with modified human genes in hopes of vanquishing the disease.

In the US, the first planned trials to use CRISPR in people still have not gotten under way. But in China, things appear to be moving relatively quickly.

Last fall, a team at Sichuan Universitys West China Hospital used CRISPR for the first time on an adult with lung cancer. In the new trial, reported by The Wall Street Journal, altered genes were injected into a patient with a rare type of head and neck cancer, called nasopharyngeal carcinoma, at Nanjing Universitys Nanjing Drum Tower Hospital.

The aim is to use CRISPR, which allows scientists to snip out pieces of DNA with greater ease than older gene-editing techniques, to suppress the activity of a gene preventing the patients body from effectively fighting the disease. On Friday, the university announced that the first patient had received an infusion of altered cells, which are taken from their body and altered in a lab before being injected back in.

In all, 20 patients with gastric cancer, nasopharyngeal carcinoma and lymphoma are expected to participate in the trial. Its first phase is expected to conclude next year.

The other Chinese trial, in which scientists modified immune cells to attack lung cancer in 11 patients, expects to release results this year, according to the Journal.

The first US human CRISPR trial is slated to begin this summerat the University of Pennsylvania, after receiving a regulatory stamp of approval to proceed last year. In that trial, scientists plan to genetically alter patients immune cells to attack three different kinds of cancer.

Clearly, a race to cure cancer with CRISPR is underfoot. And right now at least, China seems to be winning.

[Wall Street Journal]

Read the rest here:

China Is Racing Ahead of the US in the Quest to Cure Cancer With CRISPR - Gizmodo

Asian Scientist Magazine

Another CRISPR Trial Begins GenomeWeb Researchers in China have embarked on a new trial using the gene-editing tool CRISPR/Cas9 to modify human genes to treat cancer patients, the Wall Street Journal reports. For this trial, researchers at Nanjing University have injected the first set of ... China Is Racing Ahead of the US in the Quest to Cure Cancer With CRISPRGizmodo CRISPR Used To Modify Multiple Genes In Rice | Asian Scientist ...Asian Scientist Magazine Insider Selling: Crispr Therapeutics AG (CRSP) Insider Sells ...Transcript Daily PharmiWeb.com (press release) -Market Exclusive -Wall Street Journal (subscription) all 11 news articles »

Follow this link:

Another CRISPR Trial Begins - GenomeWeb

We have anti-arthritis drugs. What we lack is the ability to deploy them when and where they are needed in the body. The drugs would be far more effective, and occasion fewer side effects, if they were to appear only in response to inflammation, and only in the joints. If the drugs could be delivered so painstakinglyso smartlythey wouldnt have to be administered systemically.

Although conventional drug delivery systems may be unable to respond to arthritic flares with such adroitness, cells may have better luckif they are suitably modified. Stem cells, for example, have been rewired by means of gene-editing technology to fight arthritis. These stem cells, known as SMART cells (Stem cells Modified for Autonomous Regenerative Therapy), develop into cartilage cells that produce a biologic anti-inflammatory drug. Ideally, the new cartilage cells will replace arthritic cartilage, and the biologic will protect against chronic inflammation, preserving joints and other tissues.

SMART cells of this sort were prepared by scientists based at Washington University School of Medicine in St. Louis. The scientists initially worked with skin cells taken from the tails of mice and converted those cells into stem cells. Then, using the gene-editing tool CRISPR in cells grown in culture, they removed a key gene in the inflammatory process and replaced it with a gene that releases a biologic drug that combats inflammation.

Details of this work appeared April 27 in the journal Stem Cell Reports, in an article entitled Genome Engineering of Stem Cells for Autonomously Regulated, Closed-Loop Delivery of Biologic Drugs. The article describes how modified stem cells grew into cartilage and produced cartilage tissue. The engineered cartilage, the scientists reported, was protected from inflammation.

Using the CRISPR/Cas9 genome-engineering system, we created stem cells that antagonize IL-1- [interleukin-1] or TNF-- [tumor necrosis factor-] mediated inflammation in an autoregulated, feedback-controlled manner, wrote the authors of the Stem Cell Reports article. Our results show that genome engineering can be used successfully to rewire endogenous cell circuits to allow for prescribed input/output relationships between inflammatory mediators and their antagonists, providing a foundation for cell-based drug delivery or cell-based vaccines via a rapidly responsive, autoregulated system.

Many current drugs used to treat arthritisincluding Enbrel (etanercept), Humira (adalimumab), and Remicade (infliximab)attack TNF-, an inflammation-promoting molecule. But the problem with these drugs is that they are given systemically rather than targeted to joints. As a result, they interfere with the immune system throughout the body and can make patients susceptible to side effects such as infections.

"We want to use our gene-editing technology as a way to deliver targeted therapy in response to localized inflammation in a joint, as opposed to current drug therapies that can interfere with the inflammatory response through the entire body," said Farshid Guilak, Ph.D., the paper's senior author and a professor of orthopedic surgery at Washington University School of Medicine. "If this strategy proves to be successful, the engineered cells only would block inflammation when inflammatory signals are released, such as during an arthritic flare in that joint."

Dr. Guilak's team encoded the stem/cartilage cells with genes that made the cells light up when responding to inflammation, so the scientists easily could determine when the cells were responding. Recently, the team began testing the engineered stem cells in mouse models of rheumatoid arthritis and other inflammatory diseases.

If the work can be replicated in animals and then developed into a clinical therapy, the engineered cells or cartilage grown from stem cells would respond to inflammation by releasing a biologic drugthe TNF- inhibitorthat would protect the synthetic cartilage cells that Dr. Guilak's team created and the natural cartilage cells in specific joints.

"When these cells see TNF-, they rapidly activate a therapy that reduces inflammation," Dr. Guilak explained. "We believe this strategy also may work for other systems that depend on a feedback loop. In diabetes, for example, it's possible we could make stem cells that would sense glucose and turn on insulin in response. We are using pluripotent stem cells, so we can make them into any cell type, and with CRISPR, we can remove or insert genes that have the potential to treat many types of disorders."

View original post here:

CRISPR-SMART Cells Regenerate Cartilage, Secrete Anti-Arthritis Drug - Genetic Engineering & Biotechnology News

Current CRISPR Patent Dispute, Explained CALIFORNIA They invented CRISPR-Cas9, a gene editing tool that uses a protein found in Streptococcus bacteria to chop up and rearrange viral DNA with precision. The implications of the technology were immediately apparent, astonishing, and perhaps just a wee bit ... and more »

Excerpt from:

Current CRISPR Patent Dispute, Explained - CALIFORNIA

Boston Business Journal

CEOs of top gene-editing firms got huge compensation hikes last year Boston Business Journal It's no secret that the burgeoning field of gene-editing a method of cutting out and replacing part of a gene has generated serious buzz in the biotech world lately. New scientific tools like CRISPR/Cas9 have the potential to revolutionize the ... CRISPR Therapeutics to Present at the 42nd Annual Deutsche Bank Health Care ConferenceGlobeNewswire (press release) Intellia (NTLA), CRISPR Therapeutics (CRSP) Receive U.S. Patent for CRISPR/Cas9 Ribonucleoprotein ComplexesStreetInsider.com Intellia Therapeutics and CRISPR Therapeutics Announce U.S. ...Yahoo Finance Texas Tribune -The Cerbat Gem -Sports Perspectives all 27 news articles »

Link:

CEOs of top gene-editing firms got huge compensation hikes last year - Boston Business Journal

For the first time, scientists have demonstrated high efficiency multiplex gene editing in plants.

Asian Scientist Newsroom | April 28, 2017 | In the Lab

AsianScientist (Apr. 28, 2017) - Using Cpf1 instead of the more familiar Cas9, researchers from China have developed an easier way to edit multiple genes with CRISPR technology, demonstrating their method in rice. Their findings have been published in Molecular Plant.

Multiplex gene editing provides a powerful tool for targeting members of multigene families. Although previous studies have shown that multiplex gene editing in plants is possible with CRISPR-Cas9, the Cas9 system requires large constructs to express multiple sgRNA cassettes, which are more laborious to construct and could cause unstability and reduce transformation efficiency.

Cpf1 is a dual nuclease that not only cleaves target DNA but also processes its own CRISPR RNA. A study led by Professor Zhu Jiankangs lab at Institute of Plant Physiology and Ecology of Chinese Academy of Sciences tested FnCpf1 and LbCpf1 for single and multiplex gene editing in rice.

Researchers found that both FnCpf1 and LbCpf1 with their own mature direct repeats induce mutations in transgenic plants. The LbCpf1 system gave higher editing efficiency in all six tested target sites.

Importantly, FnCpf1 and LbCpf1 also showed robust activity in multiplex gene editing when expressed together with a single CRISPR array. It has been proved that FnCpf1 and LbCpf1 are functional when the direct repeat sequences of their CRISPR arrays are exchanged.

This study demonstrated for the first time the feasibility of high efficiency multiplex gene editing in plants using engineered CRISPR-Cpf1 with a simple short DR-guide array, which significantly simplifies multiplex gene editing in plants.

The article can be found at: Wang et al. (2017) Multiplex Gene Editing in Rice Using the CRISPR-Cpf1 System.

Source: Chinese Academy of Sciences; Photo: Shutterstock. Disclaimer: This article does not necessarily reflect the views of AsianScientist or its staff.

Original post:

CRISPR Used To Modify Multiple Genes In Rice - Asian Scientist Magazine

Clustered regularly interspaced short palindromic repeats (CRISPR, pronounced crisper[2]) are segments of prokaryotic DNA containing short, repetitive base sequences. These play a key role in a bacterial defence system,[3] and form the basis of a genome editing technology known as CRISPR-Cas9 that allows permanent modification of genes within organisms.[4] In a palindromic repeat, the sequence of nucleotides is the same in both directions. Each repetition is followed by short segments of spacer DNA from previous exposures to foreign DNA (e.g., a virus or plasmid).[5] Small clusters of cas (CRISPR-associated system) genes are located next to CRISPR sequences.

The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages[6][7][8] that provides a form of acquired immunity. RNA harboring the spacer sequence helps Cas proteins recognize and cut exogenous DNA. Other RNA-guided Cas proteins cut foreign RNA.[9] CRISPRs are found in approximately 40% of sequenced bacterial genomes and 90% of sequenced archaea.[10][note 1]

A simple version of the CRISPR/Cas system, CRISPR/Cas9, has been modified to edit genomes. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added.[11][12][13] The Cas9-gRNA complex corresponds with the CAS III crRNA complex in the above diagram.

CRISPR/Cas genome editing techniques have many potential applications, including medicine and crop seed enhancement. The use of CRISPR/Cas9-gRNA complex for genome editing[14][15] was the AAAS's choice for breakthrough of the year in 2015.[16]Bioethical concerns have been raised about the prospect of using CRISPR for germline editing.[17]

Structure of crRNA-guided E. coli Cascade complex (Cas, blue) bound to single-stranded DNA (orange).

The discovery of clustered DNA repeats began independently in three parts of the world.

The first description of what would later be called CRISPR was from Osaka University researcher Yoshizumi Ishino in 1987, who accidentally cloned part of a CRISPR together with the iap gene, the target of interest. The organization of the repeats was unusual because repeated sequences are typically arranged consecutively along DNA. The function of the interrupted clustered repeats was not known at the time.[18][19]

In 1993 researchers of Mycobacterium tuberculosis in the Netherlands published two articles about a cluster of interrupted direct repeats (DR) in this bacterium. These researchers recognized the diversity of the DR-intervening sequences among different strains of M. tuberculosis[20] and used this property to design a typing method that was named Spoligotyping, which is still in use today.[21][22]

At the same time, repeats were observed in the archaeal organisms Haloferax and Haloarcula species, and their function was studied by Francisco Mojica at the University of Alicante in Spain. Although his hypothesis turned out to be wrong, Mojica surmised at the time that the clustered repeats had a role in correctly segregating replicated DNA into daughter cells during cell division because plasmids and chromosomes with identical repeat arrays could not coexist in Haloferax volcanii. Transcription of the interrupted repeats was also noted for the first time.[22][23] By 2000, Mojica's group had identified interrupted repeats in 20 species of microbes.[24] In 2001, Mojica and Ruud Jansen, who was searching for additional interrupted repeats, proposed the acronym CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) to alleviate the confusion stemming from the numerous acronyms used to describe the sequences in the scientific literature.[23][25]

A major addition to the understanding of CRISPR came with Jansen's observation that the prokaryote repeat cluster was accompanied by a set of homologous genes that make up CRISPR-associated systems or cas genes. Four cas genes (cas 1 to 4) were initially recognized. The Cas proteins showed helicase and nuclease motifs, suggesting a role in the dynamic structure of the CRISPR loci.[26] In this publication the acronym CRISPR was coined as the universal name of this pattern. However, the CRISPR function remained enigmatic.

In 2005, three independent research groups showed that some CRISPR spacers are derived from phage DNA and extrachromosomal DNA such as plasmids.[27][28][29] In effect, the spacers are fragments of DNA gathered from viruses that previously tried to attack the cell. The source of the spacers was a sign that the CRISPR/cas system could have a role in adaptive immunity in bacteria.[1][30] All three studies proposing this idea were initially rejected by high-profile journals, but eventually appeared in other journals.[31]

The first publication[28] proposing a role of CRISPR-Cas in microbial immunity, by Mojica's group, predicted a role for the RNA transcript of spacers on target recognition in a mechanism that could be analogous to the RNA interference system used by eukaryotic cells. Koonin and colleagues extended that hypothesis by proposing mechanisms of action for the different CRISPR-Cas subtypes according to the predicted function of their proteins.[32] Others hypothesized that CRISPR sequences directed Cas enzymes to degrade viral DNA.[19][29]

Experimental work by several groups revealed the basic mechanisms of CRISPR-Cas immunity. In 2007 the first experimental evidence that CRISPR was an adaptive immune system was published.[19] A CRISPR region in Streptococcus thermophilus acquired spacers from the DNA of an infecting bacteriophage. The researchers manipulated the resistance of S. thermophilus to phage by adding and deleting spacers whose sequence matched those found in the tested phages.[33][34] In 2008, Brouns and colleagues identified a complex of Cas protein that in E. coli cut the CRISPR RNA within the repeats into spacer-containing RNA molecules, which remained bound to the protein complex. That year Marraffini and Sontheimer showed that a CRISPR sequence of S. epidermidis targeted DNA and not RNA to prevent conjugation. This finding was at odds with the proposed RNA-interference-like mechanism of CRISPR-Cas immunity, although a CRISPR-Cas system that targets foreign RNA was later found in Pyrococcus furiosus.[19][33] A 2010 study showed that CRISPR-Cas cuts both strands of phage and plasmid DNA in S. thermophilus.[35]

Researchers studied a simpler CRISPR system from Streptococcus pyogenes that relies on the protein Cas9. The Cas9 endonuclease is a four-component system that includes two small RNA molecules named CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA).[36]Jennifer Doudna and Emmanuelle Charpentier re-engineered the Cas9 endonuclease into a more manageable two-component system by fusing the two RNA molecules into a "single-guide RNA" that, when mixed with Cas9, could find and cut the DNA target specified by the guide RNA. By manipulating the nucleotide sequence of the guide RNA, the artificial Cas9 system could be programmed to target any DNA sequence for cleavage.[37] Another group of collaborators comprising iknys together with Gainas, Barrangou and Horvath showed that Cas9 from the S. thermophilus CRISPR system can also be reprogrammed to target a site of their choosing by changing the sequence of its crRNA. These advances fueled efforts to edit genomes with the modified CRISPR-Cas9 system.[22]

Feng Zhang's and George Church's groups simultaneously described genome editing in human cell cultures using CRISPR-Cas9 for the first time.[19][38][39] It has since been used in a wide range of organisms, including baker's yeast (Saccharomyces cerevisiae),[40][41][42] zebrafish (D. rerio),[43] fruit flies (Drosophila melanogaster),[44] nematodes (C. elegans),[45] plants,[46] mice,[47] monkeys[48] and human embryos.[49]

CRISPR has been modified to make programmable transcription factors that allow scientists to target and activate or silence specific genes.[50]

In 2015, the nuclease Cpf1 was discovered in the CRISPR/Cpf1 system of the bacterium Francisella novicida.[51][52] Cpf1 showed several key differences from Cas9 including: causing a 'staggered' cut in double stranded DNA as opposed to the 'blunt' cut produced by Cas9, relying on a 'T rich' PAM (providing alternate targeting sites to Cas9) and requiring only a CRISPR RNA (crRNA) for successful targeting. By contrast Cas9 requires both crRNA and a transactivating crRNA (tracrRNA)).

In the early 2000s, researchers developed zinc finger nucleases, synthetic proteins whose DNA-binding domains enable them to create double-stranded breaks in DNA at specific points. In 2010, synthetic nucleases called transcription activator-like effector nucleases (TALENs) provided an easier way to target a double-stranded break to a specific location on the DNA strand. Both zinc finger nucleases and TALENs require the creation of a custom protein for each targeted DNA sequence, which is a more difficult and time-consuming process than that for guide RNAs. CRISPRs are much easier to design because the process requires making only a short RNA sequence.[53]

The CRISPR array comprises an AT-rich leader sequence followed by short repeats that are separated by unique spacers.[54] CRISPR repeats typically range in size from 28 to 37 base pairs (bps), though there can be as few as 23 bp and as many as 55 bp.[55] Some show dyad symmetry, implying the formation of a secondary structure such as a hairpin in the RNA, while others are predicted to be unstructured. The size of spacers in different CRISPR arrays is typically 32 to 38 bp (range 21 to 72 bp).[55] New spacers can appear rapidly as part of the immune response to phage infection.[56] There are usually fewer than 50 units of the repeat-spacer sequence in a CRISPR array.[55]

Small clusters of cas genes are often located next to CRISPR repeat-spacer arrays. Collectively there are 93 cas genes that are grouped into 35 families based on sequence similarity of the encoded proteins. 11 of the 35 families form the cas core, which includes the protein families Cas1 through Cas9. A complete CRISPR-Cas locus has at least one gene belonging to the cas core.[57]

CRISPR-Cas systems fall into two classes. Class 1 systems use a complex of multiple Cas proteins to degrade foreign nucleic acids. Class 2 systems use a single large Cas protein for the same purpose. Class 1 is divided into types I, III, and IV; class 2 is divided into types II, V, and VI.[58] The 6 system types are divided into 19 subtypes.[59] Each type and most subtypes are characterized by a "signature gene" found almost exclusively in the category. Classification is also based on the complement of cas genes that are present. Most CRISPR-Cas systems have a Cas1 protein. The phylogeny of Cas1 proteins generally agrees with the classification system.[57] Many organisms contain multiple CRISPR-Cas systems suggesting that they are compatible and may share components.[60][61] The sporadic distribution of the CRISPR/Cas subtypes suggests that the CRISPR/Cas system is subject to horizontal gene transfer during microbial evolution.

CRISPR-Cas immunity is a natural process of bacteria and archaea. CRISPR-Cas prevents bacteriophage infection, conjugation and natural transformation by degrading foreign nucleic acids that enter the cell.[33]

When a microbe is invaded by a virus, the first stage of the immune response is to capture viral DNA and insert it into a CRISPR locus in the form of a spacer. Cas1 and Cas2 are found in all three types of CRISPR-Cas immune systems, which indicates that they are involved in spacer acquisition. Mutation studies confirmed this hypothesis, showing that removal of cas1 or cas2 stopped spacer acquisition, without affecting CRISPR immune response.[70][71][72][73][74]

Multiple Cas1 proteins have been characterised and their structures resolved.[75][76][77] Cas1 proteins have diverse amino acid sequences. However, their crystal structures are similar and all purified Cas1 proteins are metal-dependent nucleases/integrases that bind to DNA in a sequence-independent manner.[60] Representative Cas2 proteins have been characterised and possess either (single strand) ssRNA-[78] or (double strand) dsDNA-[79][80] specific endoribonuclease activity.

In the I-E system of E. coli Cas1 and Cas2 form a complex where a Cas2 dimer bridges two Cas1 dimers.[81] In this complex Cas2 performs a non-enzymatic scaffolding role,[81] binding double-stranded fragments of invading DNA, while Cas1 binds the single-stranded flanks of the DNA and catalyses their integration into CRISPR arrays.[82][83][84]

Bioinformatic analysis of regions of phage genomes that were excised as spacers (termed protospacers) revealed that they were not randomly selected but instead were found adjacent to short (3 5 bp) DNA sequences termed protospacer adjacent motifs (PAM). Analysis of CRISPR-Cas systems showed PAMs to be important for type I and type II, but not type III systems during acquisition.[29][85][86][87][88][89] In type I and type II systems, protospacers are excised at positions adjacent to a PAM sequence, with the other end of the spacer cut using a ruler mechanism, thus maintaining the regularity of the spacer size in the CRISPR array.[90][91] The conservation of the PAM sequence differs between CRISPR-Cas systems and appears to be evolutionarily linked to Cas1 and the leader sequence.[89][92]

New spacers are added to a CRISPR array in a directional manner,[27] occurring preferentially,[56][85][86][93][94] but not exclusively, adjacent[88][91] to the leader sequence. Analysis of the type I-E system from E. coli demonstrated that the first direct repeat adjacent to the leader sequence, is copied, with the newly acquired spacer inserted between the first and second direct repeats.[73][90]

The PAM sequence appears to be important during spacer insertion in type I-E systems. That sequence contains a strongly conserved final nucleotide (nt) adjacent to the first nt of the protospacer. This nt becomes the final base in the first direct repeat.[74][95][96] This suggests that the spacer acquisition machinery generates single-stranded overhangs in the second-to-last position of the direct repeat and in the PAM during spacer insertion. However, not all CRISPR-Cas systems appear to share this mechanism as PAMs in other organisms do not show the same level of conservation in the final position.[92] It is likely that in those systems, a blunt end is generated at the very end of the direct repeat and the protospacer during acquisition.

Analysis of Sulfolobus solfataricus CRISPRs revealed further complexities to the canonical model of spacer insertion, as one of its six CRISPR loci inserted new spacers randomly throughout its CRISPR array, as opposed to inserting closest to the leader sequence.[91]

Multiple CRISPRs contain many spacers to the same phage. The mechanism that causes this phenomenon was discovered in the type I-E system of E. coli. A significant enhancement in spacer acquisition was detected where spacers already target the phage, even mismatches to the protospacer. This priming requires the Cas proteins involved in both acquisition and interference to interact with each other. Newly acquired spacers that result from the priming mechanism are always found on the same strand as the priming spacer.[74][95][96] This observation led to the hypothesis that the acquisition machinery slides along the foreign DNA after priming to find a new protospacer.[96]

CRISPR-RNA (crRNA), which later guides the Cas nuclease to the target during the interference step, must be generated from the CRISPR sequence. The crRNA is initially transcribed as part of a single long transcript encompassing much of the CRISPR array.[5] This transcript is then cleaved by Cas proteins to form crRNAs. The mechanism to produce crRNAs differs among CRISPR-Cas systems. In type I-E and type I-F systems, the proteins Cas6e and Cas6f respectively, recognise stem-loops[97][98][99] created by the pairing of identical repeats that flank the crRNA.[100] These Cas proteins cleave the longer transcript at the edge of the paired region, leaving a single crRNA along with a small remnant of the paired repeat region.

Type III systems also use Cas6, however their repeats do not produce stem-loops. Cleavage instead occurs by the longer transcript wrapping around the Cas6 to allow cleavage just upstream of the repeat sequence.[101][102][103]

Type II systems lack the Cas6 gene and instead utilize RNaseIII for cleavage. Functional type II systems encode an extra small RNA that is complementary to the repeat sequence, known as a trans-activating crRNA (tracrRNA).[71] Transcription of the tracrRNA and the primary CRISPR transcript results in base pairing and the formation of dsRNA at the repeat sequence, which is subsequently targeted by RNaseIII to produce crRNAs. Unlike the other two systems the crRNA does not contain the full spacer, which is instead truncated at one end.[66]

CrRNAs associate with Cas proteins to form ribonucleotide complexes that recognize foreign nucleic acids. CrRNAs show no preference between the coding and non-coding strands, which is indicative of an RNA-guided DNA-targeting system.[8][35][70][74][104][105][106] The type I-E complex (commonly referred to as Cascade) requires five Cas proteins bound to a single crRNA.[107][108]

During the interference stage in type I systems the PAM sequence is recognized on the crRNA-complementary strand and is required along with crRNA annealing. In type I systems correct base pairing between the crRNA and the protospacer signals a conformational change in Cascade that recruits Cas3 for DNA degradation.

Type II systems rely on a single multifunctional protein, Cas9, for the interference step.[66]Cas9 requires both the crRNA and the tracrRNA to function and cleaves DNA using its dual HNH and RuvC/RNaseH-like endonuclease domains. Basepairing between the PAM and the phage genome is required in type II systems. However, the PAM is recognized on the same strand as the crRNA (the opposite strand to type I systems).

Type III systems, like type I require six or seven Cas proteins binding to crRNAs.[109][110] The type III systems analysed from S. solfataricus and P. furiosus both target the mRNA of phages rather than phage DNA genome,[61][110] which may make these systems uniquely capable of targeting RNA-based phage genomes.[60]

The mechanism for distinguishing self from foreign DNA during interference is built into the crRNAs and is therefore likely common to all three systems. Throughout the distinctive maturation process of each major type, all crRNAs contain a spacer sequence and some portion of the repeat at one or both ends. It is the partial repeat sequence that prevents the CRISPR-Cas system from targeting the chromosome as base pairing beyond the spacer sequence signals self and prevents DNA cleavage.[111] RNA-guided CRISPR enzymes are classified as type V restriction enzymes.

A bioinformatic study showed that CRISPRs are evolutionarily conserved and cluster into related types. Many show signs of a conserved secondary structure.[100]

CRISPR/Cas can immunize bacteria against certain phages and thus halt transmission. For this reason, Koonin described CRISPR/Cas as a Lamarckian inheritance mechanism.[112] However, this was disputed by a critic who noted, "We should remember [Lamarck] for the good he contributed to science, not for things that resemble his theory only superficially. Indeed, thinking of CRISPR and other phenomena as Lamarckian only obscures the simple and elegant way evolution really works".[113]

Analysis of CRISPR sequences revealed coevolution of host and viral genomes.[114] Cas9 proteins are highly enriched in pathogenic and commensal bacteria. CRISPR/Cas-mediated gene regulation may contribute to the regulation of endogenous bacterial genes, particularly during interaction with eukaryotic hosts. For example, Francisella novicida uses a unique, small, CRISPR/Cas-associated RNA (scaRNA) to repress an endogenous transcript encoding a bacterial lipoprotein that is critical for F. novicida to dampen host response and promote virulence.[115]

The basic model of CRISPR evolution is newly incorporated spacers driving phages to mutate their genomes to avoid the bacterial immune response, creating diversity in both the phage and host populations. To fight off a phage infection, the sequence of the CRISPR spacer must correspond perfectly to the sequence of the target phage gene. Phages can continue to infect their hosts given point mutations in the spacer.[111] Similar stringency is required in PAM or the bacterial strain remains phage sensitive.[86][111]

A study of 124 S. thermophilus strains showed that 26% of all spacers were unique and that different CRISPR loci showed different rates of spacer acquisition.[85] Some CRISPR loci evolve more rapidly than others, which allowed the strains' phylogenetic relationships to be determined. A comparative genomic analysis showed that E. coli and S. enterica evolve much more slowly than S. thermophilus. The latter's strains that diverged 250 thousand years ago still contained the same spacer complement.[116]

Metagenomic analysis of two acid mine drainage biofilms showed that one of the analyzed CRISPRs contained extensive deletions and spacer additions versus the other biofilm, suggesting a higher phage activity/prevalence in one community than the other.[56] In the oral cavity, a temporal study determined that 7-22% of spacers were shared over 17 months within an individual while less than 2% were shared across individuals.[94]

From the same environment a single strain was tracked using PCR primers specific to its CRISPR system. Broad-level results of spacer presence/absence showed significant diversity. However, this CRISPR added 3 spacers over 17 months,[94] suggesting that even in an environment with significant CRISPR diversity some loci evolve slowly.

CRISPRs were analysed from the metagenomes produced for the human microbiome project.[117] Although most were body-site specific, some within a body site are widely shared among individuals. One of these loci originated from streptococcal species and contained ~15,000 spacers, 50% of which were unique. Similar to the targeted studies of the oral cavity, some showed little evolution over time.[117]

CRISPR evolution was studied in chemostats using S. thermophilus to directly examine spacer acquisition rates. In one week, S. thermophilus strains acquired up to three spacers when challenged with a single phage.[118] During the same interval the phage developed single nucleotide polymorphisms that became fixed in the population, suggesting that targeting had prevented phage replication absent these mutations.[118]

Another S. thermophilus experiment showed that phages can infect and replicate in hosts that have only one targeting spacer. Yet another showed that sensitive hosts can exist in environments with high phage titres.[119] The chemostat and observational studies suggest many nuances to CRISPR and phage (co)evolution.

CRISPRs are widely distributed among bacteria and archaea[64] and show some sequence similarities.[100] Their most notable characteristic is their repeating spacers and direct repeats. This characteristic makes CRISPRs easily identifiable in long sequences of DNA, since the number of repeats decreases the likelihood of a false positive match. Three programs used for CRISPR repeat identification search for regularly interspaced repeats in long sequences: CRT,[120] PILER-CR[121] and CRISPRfinder.[122]

Analysis of CRISPRs in metagenomic data is more challenging, as CRISPR loci do not typically assemble, due to their repetitive nature or through strain variation, which confuses assembly algorithms. Where many reference genomes are available, polymerase chain reaction (PCR) can be used to amplify CRISPR arrays and analyse spacer content.[85][94][123][124][125] However, this approach yields information only for specifically targeted CRISPRs and for organisms with sufficient representation in public databases to design reliable polymerase chain reaction (PCR) primers.

The alternative is to extract and reconstruct CRISPR arrays from shotgun metagenomic data. This is computationally more difficult, particularly with second generation sequencing technologies (e.g. 454, Illumina), as the short read lengths prevent more than two or three repeat units appearing in a single read. CRISPR identification in raw reads has been achieved using purely de novo identification[126] or by using direct repeat sequences in partially assembled CRISPR arrays from contigs (overlapping DNA segments that together represent a consensus region of DNA)[117] and direct repeat sequences from published genomes[127] as a hook for identifying direct repeats in individual reads.

Another way for bacteria to defend against phage infection is by having chromosomal islands. A subtype of chromosomal islands called phage-inducible chromosomal island (PICI) is excised from a bacterial chromosome upon phage infection and can inhibit phage replication.[128] The mechanisms that induce PICI excision and how PICI inhibits phage replication are not well understood. One study showed that lytic ICP1 phage, which specifically targets Vibrio cholerae serogroup O1, has acquired a CRISPR/Cas system that targets a V. cholera PICI-like element. The system has 2 CRISPR loci and 9 Cas genes. It seems to be homologous to the 1-F system found in Yersinia pestis. Moreover, like the bacterial CRISPR/Cas system, ICP1 CRISPR/Cas can acquire new sequences, which allows phage and host to co-evolve.[129]

By the end of 2014 some 600 research papers had been published that mentioned CRISPR.[130] The technology had been used to functionally inactivate genes in human cell lines and cells, to study Candida albicans, to modify yeasts used to make biofuels and to genetically modify crop strains.[130] CRISPR can also be used to change mosquitos so they cannot transmit diseases such as malaria.[131]

CRISPR-based re-evaluations of claims for gene-disease relationships have led to the discovery of potentially important anomalies[132]

CRISPR/Cas9 genome editing is carried out with a Type II CRISPR system. When utilized for genome editing, this system includes Cas9, crRNA, tracrRNA along with an optional section of DNA repair template that is utilized in either Non-Homologous End Joining (NHEJ) or Homology Directed Repair (HDR).

CRISPR/Cas9 often employs a plasmid to transfect the target cells.[135] The main components of this plasmid are displayed in the image and listed in the table. The crRNA needs to be designed for each application as this is the sequence that Cas9 uses to identify and directly bind to the cell's DNA. The crRNA must bind only where editing is desired. The repair template is designed for each application, as it must overlap with the sequences on either side of the cut and code for the insertion sequence.

Multiple crRNAs and the tracrRNA can be packaged together to form a single-guide RNA (sgRNA).[136] This sgRNA can be joined together with the Cas9 gene and made into a plasmid in order to be transfected into cells.

CRISPR/Cas9 offers a high degree of fidelity and relatively simple construction. It depends on two factors for its specificity: the target sequence and the PAM. The target sequence is 20 bases long as part of each CRISPR locus in the crRNA array.[135] A typical crRNA array has multiple unique target sequences. Cas9 proteins select the correct location on the host's genome by utilizing the sequence to bond with base pairs on the host DNA. The sequence is not part of the Cas9 protein and as a result is customizable and can be independently synthesized.[137][138]

The PAM sequence on the host genome is recognized by Cas9. Cas9 cannot be easily modified to recognize a different PAM sequence. However this is not too limiting as it is a short sequence and nonspecific (e.g. the SpCas9 PAM sequence is 5'-NGG-3' and in the human genome occurs roughly every 8 to 12 base pairs).[135]

Once these have been assembled into a plasmid and transfected into cells the Cas9 protein with the help of the crRNA finds the correct sequence in the host cell's DNA and depending on the Cas9 variant creates a single or double strand break in the DNA.[139]

Properly spaced single strand breaks in the host DNA can trigger homology directed repair, which is less error prone than the non-homologous end joining that typically follows a double strand break. Providing a DNA repair template allows for the insertion of a specific DNA sequence at an exact location within the genome. The repair template should extend 40 to 90 base pairs beyond the Cas9 induced DNA break.[135] The goal is for the cell's HDR process to utilize the provided repair template and thereby incorporate the new sequence into the genome. Once incorporated, this new sequence is now part of the cell's genetic material and passes into its daughter cells.

Many online tools are available to aid in designing effective sgRNA sequences.[140]

Scientists can use viral or non-viral systems for delivery of the Cas9 and sgRNA into target cells. Electroporation of DNA, RNA or ribonucleocomplexes is the most common and cheapest system. This technique was used to edit CXCR4 and PD-1, knocking in new sequences to replace specific genetic "letters" in these proteins. The group was then able to sort the cells, using cell surface markers, to help identify successfully edited cells.[141] Deep sequencing of a target site confirmed that knock-in genome modifications had occurred with up to 20% efficiency, which accounted for up to approximately one-third of total editing events.[142] However, hard-to-transfect cells (stem cells, neurons, hematopoietic cells, etc.) require more efficient delivery systems such as those based on lentivirus (LVs), adenovirus (AdV) and adeno-associated virus (AAV).

CRISPRs have been used to cut five[34] to 62 genes at once: pig cells have been engineered to inactivate all 62 Porcine Endogenous Retroviruses in the pig genome, which eliminated transinfection from the pig to human cells in culture.[143] CRISPR's low cost compared to alternatives is widely seen as revolutionary.[11][12]

Selective engineered redirection of the CRISPR/Cas system was first demonstrated in 2012 in:[144][145]

Several variants of CRISPR/Cas9 allow gene activation or genome editing with an external trigger such as light or small molecules.[148][149][150] These include photoactivatable CRISPR systems developed by fusing light-responsive protein partners with an activator domain and a dCas9 for gene activation,[151][152] or fusing similar light responsive domains with two constructs of split-Cas9,[153][154] or by incorporating caged unnatural amino acids into Cas9,[155] or by modifying the guide RNAs with photocleavable complements for genome editing.[156]

Methods to control genome editing with small molecules include an allosteric Cas9, with no detectable background editing, that will activate binding and cleavage upon the addition of 4-hydroxytamoxifen (4-HT),[148] 4-HT responsive intein-linked Cas9s[157] or a Cas9 that is 4-HT responsive when fused to four ERT2 domains.[158] Intein-inducible split-Cas9 allows dimerization of Cas9 fragments[159] and Rapamycin-inducible split-Cas9 system developed by fusing two constructs of split Cas9 with FRB and FKBP fragments.[160] Furthermore, other studies have shown to induce transcription of Cas9 with a small molecule, doxycyline.[161][162] Small molecules can also be used to improve Homology Directed Repair (HDR),[163] often by inhibiting the Non-Homologous End Joining (NHEJ) pathway.[164] These systems allow conditional control of CRISPR activity for improved precision, efficiency and spatiotemporal control.

Using "dead" versions of Cas9 (dCas9) eliminates CRISPR's DNA-cutting ability, while preserving its ability to target desirable sequences. Multiple groups added various regulatory factors to dCas9s, enabling them to turn almost any gene on or off or adjust its level of activity.[165] Like RNAi, CRISPR interference (CRISPRi) turns off genes in a reversible fashion by targeting, but not cutting a site. The targeted site is methylated, epigenetically modifying the gene. This modification inhibits transcription. Cas9 is an effective way of targeting and silencing specific genes at the DNA level.[166] In bacteria, the presence of Cas9 alone is enough to block transcription. For mammalian applications, a section of protein is added. Its guide RNA targets regulatory DNA sequences called promoters that immediately precede the target gene.[34]

Cas9 was used to carry synthetic transcription factors that activated specific human genes. The technique achieved a strong effect by targeting multiple CRISPR constructs to slightly different locations on the gene's promoter.[34]

In 2016 researchers demonstrated that CRISPR from an ordinary mouth bacterium could be used to edit RNA. The researchers searched databases containing hundreds of millions of genetic sequences for those that resembled Crispr genes. They considered the fusobacteria Leptotrichia shahii. It had a group of genes that resembled CRISPR genes, but with important differences. When the researchers equipped other bacteria with these genes, which they called C2c2, they found that the organisms gained a novel defense.[167]

Many viruses encode their genetic information in RNA rather than DNA that they repurpose to make new viruses. HIV and poliovirus are such viruses. Bacteria with C2c2 make molecules that can dismember RNA, destroying the virus. Tailoring these genes opened any RNA molecule to editing.[167]

CRISPR simplifies creation of animals for research that mimic disease or show what happens when a gene is knocked down or mutated. CRISPR may be used at the germline level to create animals where the gene is changed everywhere, or it may be targeted at non-germline cells.[168][169][170]

CRISPR can be utilized to create human cellular models of disease. For instance, applied to human pluripotent stem cells CRISPR introduced targeted mutations in genes relevant to polycystic kidney disease (PKD) and focal segmental glomerulosclerosis (FSG).[171] These CRISPR-modified pluripotent stem cells were subsequently grown into human kidney organoids that exhibited disease-specific phenotypes. Kidney organoids from stem cells with PKD populations formed large, translucent cyst structures from kidney tubules. Kidney organoids with mutations in a gene linked to FSG developed junctional defects between podocytes, the filtering cells affected in that disease. Importantly, these disease phenotypes were absent in control organoids of identical genetic background, but lacking the CRISPR modifications.[171]

A similar approach was taken to model long QT syndrome in cardiomyocytes derived from pluripotent stem cells.[172] These CRISPR-generated cellular models, with isogenic controls, provide a new way to study human disease and test drugs.

In 2003 evolutionary biologist Austin Burt envisioned attaching a gene that coded for a desired trait to "selfish" DNA elements that could copy themselves from one chromosome position to another. That would bias daughter cells to inherit it, quickly spreading it throughout a population. In 2015 a U.S. team used CRISPR to create a "mutagenic chain reaction" that drove a pigmentation trait in lab-grown Drosophila to the next generation with 97% efficiency. With another research group they created a gene drive in mosquitoes that spread genes that prevented the insects from harboring malaria parasites. Weeks later, the team reported a second drive with genes that rendered female mosquitoes infertile and could quickly wipe out a population.[165]

CRISPR/Cas-based "RNA-guided nucleases" can be used to target virulence factors, genes encoding antibiotic resistance and other medically relevant sequences of interest. This technology thus represents a novel form of antimicrobial therapy and a strategy by which to manipulate bacterial populations.[173][174] Some of the affected genes are tied to human diseases, including those involved in muscle differentiation, cancer, inflammation and fetal hemoglobin.[34]

Research suggests that CRISPR is an effective way to limit replication of multiple herpesviruses. It was able to eradicate viral DNA in the case of Epstein-Barr virus (EBV). Anti-herpesvirus CRISPRs have promising applications such as removing cancer-causing EBV from tumor cells, helping rid donated organs for immunocompromised patients of viral invaders, or preventing cold sore outbreaks and recurrent eye infections by blocking HSV-1 reactivation. As of August 2016, these were awaiting testing.[175] CRISPR is being appied to develop tissue-based treatments for cancer and other diseases.[165][176]

CRISPR may revive the concept of transplanting animal organs into people. Retroviruses present in animal genomes could harm transplant recipients. In 2015 a team eliminated 62 copies of a retrovirus's DNA from the pig genome.[165]

CRISPR may have applications in tissue engineering and regenerative medicine, such as by creating human blood vessels that lack expression of MHC class II proteins, which often cause transplant rejection.[177]

In 2015, multiple studies attempted to systematically disable each individual human gene, in an attempt to identify which genes were essential to human biology. Between 1,600 and 1,800 genes passed this testof the 20,000 or so known human genes. Such genes are more strongly activated, and unlikely to carry disabling mutations. They are more likely to have indispensable counterparts in other species. They build proteins that unite to form larger collaborative complexes. The studies also catalogued the essential genes in four cancer-cell lines and identified genes that are expendable in healthy cells, but crucial in specific tumor types and drugs that could target these rogue genes.[178]

The specific functions of some 18 percent of the essential genes are unidentified. In one 2015 targeting experiment, disabling individual genes in groups of cells attempted to identify those involved in resistance to a melanoma drug. Each such gene manipulation is itself a separate "drug", potentially opening the entire genome to CRISPR-based regulation.[165]

Unenriched sequencing libraries often have abundant undesired sequences. Cas9 can specifically deplete the undesired sequences with double strand breakage with up to 99% efficiency and without significant off-target effects as seen with restriction enzymes. Treatment with Cas9 can deplete abundant rRNA while increasing pathogen sensitivity in RNA-seq libraries.[179]

As of December 2014, patent rights to CRISPR were contested. Several companies formed to develop related drugs and research tools.[180] As companies ramp up financing, doubts as to whether CRISPR can be quickly monetized were raised.[181] In February 2017 the US patent office ruled on a patent interference case brought by University of California with respect to patents issued to the Broad Institute, and found that the Broad patents, with claims covering the application of CRISPR/cas9 in eukaryotic cells, were distinct from the inventions claimed by University of California.[182][183][184]

As of November 2013, SAGE Labs (now part of Horizon Discovery group) had exclusive rights from one of those companies to produce and sell genetically engineered rats and non-exclusive rights for mouse and rabbit models.[185] By 2015[update], Thermo Fisher Scientific had licensed intellectual property from ToolGen to develop CRISPR reagent kits.[186]

At least four labs in the US, labs in China and the UK, and a US biotechnology company called Ovascience announced plans or ongoing research to apply CRISPR to human embryos.[187] Scientists, including a CRISPR co-inventor, urged a worldwide moratorium on applying CRISPR to the human germline, especially for clinical use. They said "scientists should avoid even attempting, in lax jurisdictions, germline genome modification for clinical application in humans" until the full implications "are discussed among scientific and governmental organizations".[49][188] These scientists support basic research on CRISPR and do not see CRISPR as developed enough for any clinical use in making heritable changes to humans.[189]

In April 2015, Chinese scientists reported results of an attempt to alter the DNA of non-viable human embryos using CRISPR to correct a mutation that causes beta thalassemia, a lethal heritable disorder.[190][191] The study had previously been rejected by both Nature and Science in part because of ethical concerns.[192] The experiments resulted in changing only some genes, and had off-target effects on other genes. The researchers stated that CRISPR is not ready for clinical application in reproductive medicine.[192] In April 2016 Chinese scientists were reported to have made a second unsuccessful attempt to alter the DNA of non-viable human embryos using CRISPR - this time to alter the CCR5 gene to make the embryo HIV resistant.[193]

In December 2015, an International Summit on Human Gene Editing took place in Washington under the guidance of David Baltimore. Members of national scientific academies of America, Britain and China discussed the ethics of germline modification. They agreed to support basic and clinical research under appropriate legal and ethical guidelines. A specific distinction was made between somatic cells, where the effects of edits are limited to a single individual, versus germline cells, where genome changes could be inherited by future generations. Heritable modifications could have unintended and far-reaching consequences for human evolution, genetically (e.g. gene/environment interactions) and culturally (e.g. Social Darwinism). Altering of gametocytes and embryos to generate inheritable changes in humans was defined to be irresponsible. The group agreed to initiate an international forum to address such concerns and harmonize regulations across countries.[194]

Policy regulations for the CRISPR/cas9 system vary around the globe. In February 2016, British scientists were given permission by regulators to genetically modify human embryos by using CRISPR-Cas9 and related techniques. However, researchers were forbidden from implanting the embryos and the embryos were to be destroyed after seven days.[195]

The US has an elaborate, interdepartmental regulatory system to evaluate new genetically modified foods and crops. For example, the Agriculture Risk Protection Act of 2000 gives the USDA the authority to oversee the detection, control, eradication, suppression, prevention, or retardation of the spread of plant pests or noxious weeds to protect the agriculture, environment and economy of the US. The act regulates any genetically modified organism that utilizes the genome of a predefined 'plant pest' or any plant not previously categorized.[196] In 2015, Yang successfully deactivated 16 specific genes in the white button mushroom. Since he had not added any foreign DNA to his organism, the mushroom could not be regulated under by the USDA under Section 340.2.[197] Yang's white button mushroom was the first organism genetically modified with the Crispr/cas9 protein system to pass US regulation.[198] In 2016, the USDA sponsored a committee to consider future regulatory policy for upcoming genetic modification techniques. With the help of the US National Academies of Sciences, Engineering and Medicine, special interests groups met on April 15 to contemplate the possible advancements in genetic engineering within the next 5 years and potential policy regulations that would need to come into play.[199] With the emergence of rogue genetic engineers employing the technology, the FDA has begun issuing new regulations.[200]

In 2012 and 2013, CRISPR was a runner-up in Science Magazine's Breakthrough of the Year award. In 2015, it was the winner of that award.[165] CRISPR was named as one of MIT Technology Review's 10 breakthroughs technologies in 2014 and 2016.[201][202]

CRISPR-DR2: Secondary structure taken from the Rfam database. Family RF01315.

CRISPR-DR5: Secondary structure taken from the Rfam database. Family RF011318.

CRISPR-DR6: Secondary structure taken from the Rfam database. Family RF01319.

CRISPR-DR8: Secondary structure taken from the Rfam database. Family RF01321.

CRISPR-DR9: Secondary structure taken from the Rfam database. Family RF01322.

CRISPR-DR19: Secondary structure taken from the Rfam database. Family RF01332.

CRISPR-DR41: Secondary structure taken from the Rfam database. Family RF01350.

CRISPR-DR52: Secondary structure taken from the Rfam database. Family RF01365.

CRISPR-DR57: Secondary structure taken from the Rfam database. Family RF01370.

CRISPR-DR65: Secondary structure taken from the Rfam database. Family RF01378.

Read the rest here:

CRISPR - Wikipedia

The development of efficient and reliable ways to make precise, targeted changes to the genome of living cells is a long-standing goal for biomedical researchers. Recently, a new tool based on a bacterial CRISPR-associated protein-9 nuclease (Cas9) from Streptococcus pyogenes has generated considerable excitement (1). This follows several attempts over the years to manipulate gene function, including homologous recombination (2) and RNA interference (RNAi) (3). RNAi, in particular, became a laboratory staple enabling inexpensive and high-throughput interrogation of gene function (4, 5), but it is hampered by providing only temporary inhibition of gene function and unpredictable off-target effects (6). Other recent approaches to targeted genome modification zinc-finger nucleases [ZFNs, (7)] and transcription-activator like effector nucleases [TALENs (8)] enable researchers to generate permanent mutations by introducing doublestranded breaks to activate repair pathways. These approaches are costly and time-consuming to engineer, limiting their widespread use, particularly for large scale, high-throughput studies.

The functions of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes are essential in adaptive immunity in select bacteria and archaea, enabling the organisms to respond to and eliminate invading genetic material. These repeats were initially discovered in the 1980s in E. coli (9), but their function wasnt confirmed until 2007 by Barrangou and colleagues, who demonstrated that S. thermophilus can acquire resistance against a bacteriophage by integrating a genome fragment of an infectious virus into its CRISPR locus (10).

Three types of CRISPR mechanisms have been identified, of which type II is the most studied. In this case, invading DNA from viruses or plasmids is cut into small fragments and incorporated into a CRISPR locus amidst a series of short repeats (around 20 bps). The loci are transcribed, and transcripts are then processed to generate small RNAs (crRNA CRISPR RNA), which are used to guide effector endonucleases that target invading DNA based on sequence complementarity (Figure 1) (11).

One Cas protein, Cas9 (also known as Csn1), has been shown, through knockdown and rescue experiments to be a key player in certain CRISPR mechanisms (specifically type II CRISPR systems). The type II CRISPR mechanism is unique compared to other CRISPR systems, as only one Cas protein (Cas9) is required for gene silencing (12). In type II systems, Cas9 participates in the processing of crRNAs (12), and is responsible for the destruction of the target DNA (11). Cas9s function in both of these steps relies on the presence of two nuclease domains, a RuvC-like nuclease domain located at the amino terminus and a HNH-like nuclease domain that resides in the mid-region of the protein (13).

To achieve site-specific DNA recognition and cleavage, Cas9 must be complexed with both a crRNA and a separate trans-activating crRNA (tracrRNA or trRNA), that is partially complementary to the crRNA (11). The tracrRNA is required for crRNA maturation from a primary transcript encoding multiple pre-crRNAs. This occurs in the presence of RNase III and Cas9 (12).

During the destruction of target DNA, the HNH and RuvC-like nuclease domains cut both DNA strands, generating double-stranded breaks (DSBs) at sites defined by a 20-nucleotide target sequence within an associated crRNA transcript (11, 14). The HNH domain cleaves the complementary strand, while the RuvC domain cleaves the noncomplementary strand.

The double-stranded endonuclease activity of Cas9 also requires that a short conserved sequence, (25 nts) known as protospacer-associated motif (PAM), follows immediately 3- of the crRNA complementary sequence (15). In fact, even fully complementary sequences are ignored by Cas9-RNA in the absence of a PAM sequence (16).

The simplicity of the type II CRISPR nuclease, with only three required components (Cas9 along with the crRNA and trRNA) makes this system amenable to adaptation for genome editing. This potential was realized in 2012 by the Doudna and Charpentier labs (11). Based on the type II CRISPR system described previously, the authors developed a simplified two-component system by combining trRNA and crRNA into a single synthetic single guide RNA (sgRNA). sgRNAprogrammed Cas9 was shown to be as effective as Cas9 programmed with separate trRNA and crRNA in guiding targeted gene alterations (Figure 2A).

To date, three different variants of the Cas9 nuclease have been adopted in genome-editing protocols. The first is wild-type Cas9, which can site-specifically cleave double-stranded DNA, resulting in the activation of the doublestrand break (DSB) repair machinery. DSBs can be repaired by the cellular Non-Homologous End Joining (NHEJ) pathway (17), resulting in insertions and/or deletions (indels) which disrupt the targeted locus. Alternatively, if a donor template with homology to the targeted locus is supplied, the DSB may be repaired by the homology-directed repair (HDR) pathway allowing for precise replacement mutations to be made (Figure 2A) (17, 18).

Cong and colleagues (1) took the Cas9 system a step further towards increased precision by developing a mutant form, known as Cas9D10A, with only nickase activity. This means it cleaves only one DNA strand, and does not activate NHEJ. Instead, when provided with a homologous repair template, DNA repairs are conducted via the high-fidelity HDR pathway only, resulting in reduced indel mutations (1, 11, 19). Cas9D10A is even more appealing in terms of target specificity when loci are targeted by paired Cas9 complexes designed to generate adjacent DNA nicks (20) (see further details about paired nickases in Figure 2B).

The third variant is a nuclease-deficient Cas9 (dCas9, Figure 2C) (21). Mutations H840A in the HNH domain and D10A in the RuvC domain inactivate cleavage activity, but do not prevent DNA binding (11, 22). Therefore, this variant can be used to sequence-specifically target any region of the genome without cleavage. Instead, by fusing with various effector domains, dCas9 can be used either as a gene silencing or activation tool (21, 2326). Furthermore, it can be used as a visualization tool. For instance, Chen and colleagues used dCas9 fused to Enhanced Green Fluorescent Protein (EGFP) to visualize repetitive DNA sequences with a single sgRNA or nonrepetitive loci using multiple sgRNAs (27).

Targeting efficiency, or the percentage of desired mutation achieved, is one of the most important parameters by which to assess a genome-editing tool. The targeting efficiency of Cas9 compares favorably with more established methods, such as TALENs or ZFNs (8). For example, in human cells, custom-designed ZFNs and TALENs could only achieve efficiencies ranging from 1% to 50% (2931). In contrast, the Cas9 system has been reported to have efficiencies up to >70% in zebrafish (32) and plants (33), and ranging from 25% in induced pluripotent stem cells (34). In addition, Zhou and colleagues were able to improve genome targeting up to 78% in one-cell mouse embryos, and achieved effective germline transmission through the use of dual sgRNAs to simultaneously target an individual gene (35).

A widely used method to identify mutations is the T7 Endonuclease I mutation detection assay (36, 37) (Figure 3). This assay detects heteroduplex DNA that results from the annealing of a DNA strand, including desired mutations, with a wildtype DNA strand (37).

Another important parameter is the incidence of off-target mutations. Such mutations are likely to appear in sites that have differences of only a few nucleotides compared to the original sequence, as long as they are adjacent to a PAM sequence. This occurs as Cas9 can tolerate up to 5 base mismatches within the protospacer region (36) or a single base difference in the PAM sequence (38). Off-target mutations are generally more difficult to detect, requiring whole-genome sequencing to rule them out completely.

Recent improvements to the CRISPR system for reducing off-target mutations have been made through the use of truncated gRNA (truncated within the crRNA-derived sequence) or by adding two extra guanine (G) nucleotides to the 5 end (28, 37). Another way researchers have attempted to minimize off-target effects is with the use of paired nickases (20). This strategy uses D10A Cas9 and two sgRNAs complementary to the adjacent area on opposite strands of the target site (Figure 2B). While this induces DSBs in the target DNA, it is expected to create only single nicks in off-target locations and, therefore, result in minimal off-target mutations.

By leveraging computation to reduce off-target mutations, several groups have developed webbased tools to facilitate the identification of potential CRISPR target sites and assess their potential for off-target cleavage. Examples include the CRISPR Design Tool (38) and the ZiFiT Targeter, Version 4.2 (39, 40).

Following its initial demonstration in 2012 (9), the CRISPR/Cas9 system has been widely adopted. This has already been successfully used to target important genes in many cell lines and organisms, including human (34), bacteria (41), zebrafish (32), C. elegans (42), plants (34), Xenopus tropicalis (43), yeast (44), Drosophila (45), monkeys (46), rabbits (47), pigs (42), rats (48) and mice (49). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA (14, 21, 29). Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations (50). A recent exciting development is the use of the dCas9 version of the CRISPR/Cas9 system to target protein domains for transcriptional regulation (26, 51, 52), epigenetic modification (25), and microscopic visualization of specific genome loci (27).

The CRISPR/Cas9 system requires only the redesign of the crRNA to change target specificity. This contrasts with other genome editing tools, including zinc finger and TALENs, where redesign of the protein-DNA interface is required. Furthermore, CRISPR/Cas9 enables rapid genome-wide interrogation of gene function by generating large gRNA libraries (51, 53) for genomic screening.

The rapid progress in developing Cas9 into a set of tools for cell and molecular biology research has been remarkable, likely due to the simplicity, high efficiency and versatility of the system. Of the designer nuclease systems currently available for precision genome engineering, the CRISPR/Cas system is by far the most user friendly. It is now also clear that Cas9s potential reaches beyond DNA cleavage, and its usefulness for genome locus-specific recruitment of proteins will likely only be limited by our imagination.

From NEB expressions Issue I, 2014 Article by Alex Reis, Ph.D., Bitesize Bio Breton Hornblower, Ph.D., Brett Robb, Ph.D.and George Tzertzinis, Ph.D., New England Biolabs, Inc.

Read the original post:

CRISPR/Cas9 and Targeted Genome Editing: A New Era in ...

News and research before you hear about it on CNBC and others. Claim your 2-week free trial to StreetInsider Premium here.

Intellia Therapeutics, Inc. (NASDAQ: NTLA) and CRISPR Therapeutics AG (NASDAQ: CRSP), two leading genome editing companies focused on the development of potentially curative therapies, announced that the United States Patent and Trademarks Office (USPTO) is expected to issue a CRISPR/Cas9 genome editing patent to Vilnius University (Vilnius). Intellia and CRISPR are nonexclusive sublicensees for a defined field of human therapeutic, prophylactic, and palliative uses (including companion diagnostics), excluding anti-fungal and anti-microbial applications.

The Vilnius patent claims are directed to CRISPR/Cas9 complexes assembled in vitro and used for site-specific modification of target DNA sequences. CRISPR/Cas9 complexes, referred to as CRISPR ribonucleoproteins or RNPs, are contemplated for use in a number of ex vivo applications in which cells, such as blood cells, may be corrected or edited outside of the body before being returned to a patient as a potential therapeutic. The patent is expected to issue on May 2, 2017 as U.S. Patent No. 9,637,739.

This new patent, together with the companies respective rights to foundational CRISPR/Cas9 intellectual property co-owned by The Regents of the University of California, University of Vienna and Dr. Emmanuelle Charpentier, provide CRISPR and Intellia with complementary rights to inventions claimed by the earliest developers in the discovery and application of CRISPR/Cas9 technology.

Intellia has a non-exclusive, royalty-free, worldwide sublicense to the Vilnius intellectual property through a 2014 license agreement with Caribou Biosciences, Inc., under which Intellia has an exclusive, worldwide sublicense to certain of Caribous developed or in-licensed CRISPR/Cas9 technology intellectual property for a defined field of human therapeutic, prophylactic, and palliative uses (including companion diagnostics), excluding anti-fungal and anti-microbial applications. Caribou has certain rights to Vilnius Universitys intellectual property through a cross-license agreement with the DuPont Company.

CRISPR acquired rights to this patent as a result of a cross-option and license agreement with Intellia which was completed in connection with the global agreement on foundational intellectual property for CRISPR/Cas9 gene editing that both companies jointly announced with the co-owners and licensors, as well as another licensee, on December 16, 2016. Under the cross-option and license agreement, CRISPR has a royalty-free worldwide sublicense to Intellias rights to the Vilnius intellectual property.

Link:

Intellia (NTLA), CRISPR Therapeutics (CRSP) Receive U.S. Patent for CRISPR/Cas9 Ribonucleoprotein Complexes - StreetInsider.com

transOMIC technologies now offers the pCLIP-dual vector system for combinatorial knockouts, where one vector expresses two gRNAs, each targeting a separate gene.

The transEDIT-dualCRISPR system was developed in collaboration with Dr. Greg Hannon ofCold Spring Harbor LaboratoryandCancer Research UKand Dr. Simon Knott ofCedars-Sinai, using a new algorithm that creates novel and superior gRNA sequences targeting the human genome.

"Our transEDIT-dualCRISPR products give researchers a superior set of genome-editing tools. These tools speed up the pace of our understanding of complex biological pathways, which helps to rapidly identify key therapeutic targets," said Blake Simmons, CEO of transOMIC technologies. "We expect the transEDIT-dualCRISPR arrayed library and our new combinatorial gene knockout kits to take our customers' research further, faster."

More information about the transEDIT-dualCRISPR arrayed library and combinatorial knockout kits can be found athttp://www.transomic.com/Products/transEdit/transEDIT-dual-CRISPR/Product-Overview.aspx

About transOMIC:Since 2012, transOMIC has been providing the international scientific community with research products that help unravel genetic complexity and give insight into gene function, ultimately providing biological understanding of disease and possibilities for therapeutics.We have an extensive offering of gene-based products to enable research scientists to perform genome editing, gene knockdown, and gene over-expression studies. Our leading-edge products are developed through ongoing collaborations with academic thought leaders.

CONTACT: Blake Simmons, 1-256-327-9513, blake@transomic.com

To view the original version on PR Newswire, visit:http://www.prnewswire.com/news-releases/transomic-technologies-launches-transedit-dual-crispr-combinatorial-gene-knockout-kits-300446303.html

SOURCE transOMIC technologies

http://www.transomic.com

Excerpt from:

transOMIC technologies Launches transEDIT-dual CRISPR ... - PR Newswire (press release)

DUBLIN--(BUSINESS WIRE)--Research and Markets has announced the addition of the "Global Crispr Market Forecast 2017-2025" report to their offering.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is fragments of prokaryotic DNA containing short redundancies of base sequences.

The Global CRISPR market by application generated revenue of $361 Million in 2016 and is anticipated to contribute $5966 Million by 2025, growing at a CAGR of 36.79% during the forecasted period of 2017-2025. The global CRISPR market is segmented on the basis of geographical analysis, product and end-user.

Increasing demand in drug discovery, late pregnancies leading to birth disorders, synthetic genes leading the way; aging genetic disorders and investment in path breaking research technology are the drivers for CRISPR Market. Drug discovery technology market plays a dominant role in boosting the CRISPR market. Genome editing has been revolutionized with the discovery of the CRISPR-CAS9 system from streptococcus pyogenes.

The possible misuse of CRISPR gene editing tool, slow growth and lack of healthcare budgets in developing countries are some of the restraints for CRISPR Market. CRISPR is linked with various applications such as Genome editing, genetic engineering etc. These applications are considered controversial due to the ethical concerns.

The global CRISPR Market covers the regions of North America, Europe and Asia Pacific. North America is the leading and has the biggest CRISPR Market; it is also anticipated that it will dominate the global cell therapy market. Europe CRISPR market is estimated to grow during the forecast period. Asia Pacific is a dominating region for CRISPR market with its applications like agriculture and animal breeding.

The major market players for CRISPR market are Editas Medicine, Crispr Therapeutics Ag, Horizon Discovery Plc., Sigma-Aldrich, Genscript, Transposagen Biopharmaceuticals, Thermo Fisher Scientific, Caribou Biosciences, Inc., and Precision Biosciences among others.

Market Determinants

Drivers

Restraints

Opportunities

Challenges

Key Topics Covered:

1. Research Scope

2. Research Methodology

3. Executive Summary

4. Market Overview

5. Analytical Overview

6. Market Determinants

7. Market Segmentation

8. Geographical Analysis

9. Company Profiling

For more information about this report visit http://www.researchandmarkets.com/research/63hp4q/global_crispr

Go here to read the rest:

Global CRISPR Market Forecast 2017-2025 - Research and Markets ... - Business Wire (press release)

Technology Networks

Quick, Sensitive Diagnostic Tests with CRISPR | Technology Networks Technology Networks Technology Networks is an internationally recognised publisher that provides access to the latest scientific news, products, research, videos and posters. and more »

Read the original:

Quick, Sensitive Diagnostic Tests with CRISPR | Technology Networks - Technology Networks

I

nventing a nonsurgical way to zap away fat is probably not the first thing that comes to mind when one thinks about the revolutionary genome-editing technique CRISPR, but maybe it should be.

A Boston dermatologist credited with developing the novelapproach to fat loss is now the owner of aprized internet domain: crispr.com.

Though perhaps not as lucrative as the technology itself, thedomain could have been advantageousforCRISPR-focusedcompanies Editas Medicine, CRISPR Therapeutics, Intellia that might have seen a clever branding opportunity. Butuntil recently, crispr.com had languished in the electronic ether for a decade, under the control of a cybersquatting computer engineer.

advertisement

According tothe Internet Corporation for Assigned Names and Numbers (ICANN), the nonprofit organization that coordinates domain names, however, CRISPR.comis now owned by Dr. Dieter Manstein.

The German-born dermatologist, who is affiliated with Massachusetts General Hospital, is best known for inventing Coolsculpting, a controlled cooling way to remove body fat, and although he also does serious research on melanoma,he does not seem to be into genome-editing for, say, acne prevention.

University of California appeals CRISPR patent setback

Manstein did not reply to interview requests, but internet records show he is a prolific buyer of domain names, with 776 registered to his Gmail account. They include some related to his profession laserskintreatments.net, lasertattoo.org, bodysculpting.com, and germanskincare.com, for example and some not, such as iwantmyson.com.

No public records indicate what Manstein paid for crispr.com, but some domain names similar to crispr.com are currentlygoing for up to five figures.An auction for crisprcas9.co (Cas9 is the enzyme used in the most common CRISPR system) starts at $2,000, with bids due May 8, while genecrispr.com and genomecrispr.com were both asking a shade under $40,000.

Experts doubt the domain name purchased by Manstein commanded anything close to this years priciest so far. 01.com, for instance, would have set you back $1,820,000, while Refi.com sold for $500,000 and Physician.com for $179,000.

Nikolay Kolev was the previous owner of CRISPR.com. Kolev, who on Twitter describes himself as a father, husband, software developer, Orthodox Christian, and Bulgarian in California, wasnt prescient when he registered crispr.com in March 2006.

Rather, he credited the domains existence to the photo-sharing site Flickr, which was popular when he first registered CRISPR.com in 2006.

I registered (or won it on an auction, I cant recall) as a Flickr-like version of crisper, he told STAT via Twitter. Yes, Flickr was cool at the time.

Like many domain name owners, Kolev didnt build a website. He did not answer questions about whether he was approached by any of the biotech companies, and none of the likely suspectsreplied to questions from STAT as to whether they ever sought to purchase it.

Domain-name brokers, however, noticed that a transaction for crispr.com was in escrow as of March 31, meaning a buyer had deposited payment with a third party and was waiting for Kolev to transfer ownership. As of this month, according to ICANN, the new owner is Manstein.

Sharon Begley can be reached at sharon.begley@statnews.com Follow Sharon on Twitter @sxbegle

Trending

Science march on Washington, billed as historic, plagued by

Science march on Washington, billed as historic, plagued by organizational turmoil

A virus, fished out of a lake, may have

A virus, fished out of a lake, may have saved a mans life and advanced

Behind the photo: How heroin took over an Ohio

Behind the photo: How heroin took over an Ohio town

Recommended

Chan, Zuckerberg boost open-access biology with grant to Cold

Chan, Zuckerberg boost open-access biology with grant to Cold Spring Harbor

The Broad Institute is testing the limits of what

The Broad Institute is testing the limits of what nonprofit means

Prize fund for new antibiotics could mean true innovation

Prize fund for new antibiotics could mean true innovation

Go here to see the original:

CRISPR.com was for sale, and you won't guess who bought it - STAT

Even since Alexander Fleming stumbled across penicillinthe first antibiotic drugscientists knew our fight with evolution was on.

Most antibiotics work by blocking biological processes that allow bacteria to thrive and multiply. With prolonged, low-dosage use, however, antibiotics become a source of pressure that forces bacteria to evolveand because these microorganisms are extremely adept at swapping and sharing bits of their DNA, when one member becomes resistant, so does most of its population.

Even more terrifying is this: because the same family of antibiotics generally act on the same biological pathways, when bacteria generate a mutation that resists one type of drug, it often renders that entire family of drugs useless.

The arms race with increasing high rates of antibiotic resistance has forced scientists to think outside the box. Although still a work in progress, teams of scientists are now working on a truly creative strategy: a pill carrying the genome-editing power tool CRISPR that instructs harmful bacteria to shred their own genes to bits.

In essence, scientists are returning CRISPR to its roots. While best known as a handy way to manipulate DNA in mice and humans, CRISPR is actually a part of the bacterias immune defense system.

Just like our immune systems can turn against ourselves, scientists are now hoping to give harmful bacteria a destructive autoimmune disease.

When optimized, a CRISPR pill could have the ability to precisely target single strains of harmful bacteria, while leaving other typesincluding beneficial bacteria in the gutintact.

First, the bad news: were rapidly losing our war on microbugs, and if things dont change were heading full throttle into an antibiotic apocalypse.

Part of the bacterias survival prowess comes from their ability to rapidly multiply. Under the right conditionsa damp, nutrition-packed human cell, for examplethe common gut bug E. Coli multiplies exponentially, doubling every thirty minutes. This gives their DNA plenty of chances to mutate and for the species to adapt.

Whats more, bacteria arent stingy about sharing their DNA. Antibiotic-resistant genes are often carried on snippets of genetic material that floats around in the bacterias innards. Microbugs can literally extend a tube out to their neighbors to inject these genetic pieces, thus sharing their resistant genes far and wide.

In what is likely the most chilling demonstration of antibacterial resistance in action, you can now watch a strain of bacteria become impervious to increasingly higher doses of an antibioticup to 1,000 times higherin just 11 days.

Obviously, heaping larger and larger doses of drugs on already weak patients isnt the solution. What we need isnt stronger drugs, but smarter drugs.

Most of our current antibiotics work in one of few ways: interfering with the bacterias DNA repair system, stopping the bacterias ability to reproduce, or weakening the bacterias cell wallsomething our cells dont haveuntil it explodes.

The downside of antibiotics is they are a sledgehammer that depletes and destroys the gut microbial community, says Dr. Jan Peter van Pijkeren at the University of Madison-Wisconsin, who is working on CRISPR-based antibiotics. You want to instead use a scalpel in order to specifically eradicate the microbe of interest.

The new CRISPR pill eschews all traditional ideas, instead relying on the bacterias mortal enemy: a type of virus called bacteriophages (or, more endearingly, phages).

Like all viruses, phages cant reproduce on their own. Instead, they constantly invade bacteria and inject viral genomes into the hosts, hoping to co-opt bacterial machinery to make armies of phage replicas.

This onslaught of foreign genetic material has spurred bacteria into developing a sophisticated defense system. When bacteria detect viral DNA, they store bits and pieces of it into their own genome to form a genetic sequence that we call CRISPRa molecular memory of the phage, so to speak.

When the bacteria detect a matching viral DNA sequence, they activate CRISPR and, together with a pair of protein scissors called Cas-9, the system destroys the viral DNA. Voila, invasion blocked.

Scientists have found that the CRISPR system doesnt cut the bacterias own DNA under normal circumstances; when it does, the result is lethal.

This spurred an ingenious idea: scientists could use phages to inject custom Trojan horses that trick the bacteria into cutting its own genes.

The idea of CRISPR-based approaches is to enact sequence-specific antimicrobial activity, placing selective pressure against genes that are bad rather than conserved bacterial targets, explains Dr. Timothy Lu at MIT.

Previously, Lus team successfully manufactured phages that carry DNA similar to antibiotic-resistant genes. Because the phage DNA was misdeemed a foreign invader, it spurred the bacterias CRISPR system into action, snipping away at its own genome and committing suicide.

Bacteria cells without the resistant gene didnt sound their alarm bells, and in turn were spared and ended up dominating the population.

Similarly, van Pijkeren is working on a CRISPR pill that contains a phage harboring bits of genomic DNA of Clostridium difficile.

C. diff is an infection that is notoriously difficult to treat, causing long-term gastrointestinal distress in nearly half a million Americans in a single year, resulting in at least 15,000 deaths. The current best treatmentwhen antibiotics failis a fecal transplant from healthy donors, but the method is still considered experimental, and long-term effects are unclear (there is, of course, also the ick factor).

Because phages are readily chewed up in our stomach acid, van Pijkeren is working on packaging them up into Lactobacillus bacteriaa common probiotic often present in yogurt.

As an initial proof-of-concept step, the team has successfully engineered Lactobacillus bacteria to produce phages that target themselves. The next step, van Pijekeren explains, is to use these probiotics as motherships that travel the gut, dispensing phages along the way to infect any nearby C. diff, and tricking them into hacking up their own DNA.

Toexploit these microbes to deliver therapeutics is very appealing because we know humans have been safely consuming them for thousands of years, says van Pijkeren.

Promises aside, scientists already see several snags that might prevent the CRISPR pill from unleashing its full power.

Part of it is the delivery vehiclethe phage. Phages are rather particular about the types of bacteria they invade, and matching courier to target will require additional research.

Scientists also worry that if not all targeted bacteria are killed or if the process isnt fast enough, some resilient members may evade the attack. Bacteria are also known to develop resistance to phage invasionwhich means that if the treatment continues, selection pressure will push the population towards resistance.

But phages arent the only way to deliver CRISPR pills, and scientists are hopeful.

The way I view it is not that we will be able to make an evolution-proof therapy, but that the genetic engineering tools will become more robust so that as evolution happens, we can rapidly develop countermeasures, says Lu.

Banner Image Credit: Shutterstock

More:

CRISPR Pill May Be Key in Fight Against Antibiotic Resistance - Singularity Hub

LSIPR and law firm HGF held a webinar yesterday focusing on CRISPR and how to navigate the IP landscape.

The two presenters from HGF, Dr Claire Irvine and Catherine Coombes, covered multiple angles on this groundbreaking technology, including the science behind it and the named inventors for the technology.

The panellists also spoke in detail about licensingin European countries, among other issues.

In the US, a CRISPR patent dispute is continuing with an appeal from the University of California (UC), Berkeley in a case against the Broad Institute of Harvard and MITs patents concerning the technology.

The Patent Trial and Appeal Board ruled in February that the Broads patents concerning CRISPR do not interfere with patent claims filed by UC.

The case was referred to in the presentation as potentially being the biggest patentability mess ever.

To watch the webinar, click here. The full presentation is also available on demand.

See the rest here:

CRISPR webinar: HGF discusses IP landscape - Life Sciences Intellectual Property Review (subscription)

CRISPR technology is a simple yet powerful tool for editing genomes. It allows researchers to easily alter DNA sequences and modify gene function. Its many potential applications include correcting genetic defects, treating and preventing the spread of diseases and improving crops. However, its promise also raises ethical concerns.

In popular usage, "CRISPR" (pronounced "crisper") is shorthand for "CRISPR-Cas9." CRISPRs are specialized stretches of DNA. The protein Cas9 (or "CRISPR-associated") is an enzyme that acts like a pair of molecular scissors, capable of cutting strands of DNA.

CRISPR technology was adapted from the natural defense mechanisms of bacteria and archaea (the domain of single-celled microorganisms). These organisms use CRISPR-derived RNA and various Cas proteins, including Cas9, to foil attacks by viruses and other foreign bodies. They do so primarily by chopping up and destroying the DNA of a foreign invader. When these components are transferred into other, more complex, organisms, it allows for the manipulation of genes, or "editing."

CRISPRs:"CRISPR" stands for "clusters of regularly interspaced short palindromic repeats." It is a specialized region of DNA with two distinct characteristics: the presence of nucleotide repeats and spacers. Repeated sequences of nucleotides the building blocks of DNA are distributed throughout a CRISPR region. Spacers are bits of DNA that are interspersed among these repeated sequences.

In the case of bacteria, the spacers are taken from viruses that previously attacked the organism. They serve as a bank of memories, which enables bacteria to recognize the viruses and fight off future attacks.

This was first demonstrated experimentally by Rodolphe Barrangou and a team of researchers at Danisco, a food ingredients company. In a2007 paperpublished in the journal Science, the researchers usedStreptococcus thermophilusbacteria, which are commonly found in yogurt and other dairy cultures, as their model. They observed that after a virus attack, new spacers were incorporated into the CRISPR region. Moreover, the DNA sequence of these spacers was identical to parts of the virusgenome. They also manipulated the spacers by taking them out or putting in new viral DNA sequences. In this way, they were able to alter the bacteria's resistance to an attack by a specific virus. Thus, the researchers confirmed that CRISPRs play a role in regulating bacterial immunity.

CRISPR RNA (crRNA):Once a spacer is incorporated and the virus attacks again, a portion of the CRISPR istranscribedand processed intoCRISPR RNA, or "crRNA." The nucleotide sequence of the CRISPR acts as a template to produce a complementary sequence of single-stranded RNA.Each crRNA consists of a nucleotide repeatand a spacer portion, according to a 2014 review by Jennifer Doudna and Emmanuelle Charpentier, published in the journal Science.

Cas9:The Cas9 protein is an enzyme that cuts foreign DNA.

The protein typically binds to two RNA molecules: crRNA and another called tracrRNA (or "trans-activating crRNA"). The two then guide Cas9 to the target site where it will make its cut. This expanse of DNA is complementary to a 20-nucleotide stretch of the crRNA.

Using two separate regions, or "domains" on its structure, Cas9 cuts both strands of the DNA double helix, making what is known as a "double-stranded break," according to the 2014 Science article.

There is a built-in safety mechanism, which ensures that Cas9 doesn't just cut anywhere in a genome. Short DNA sequences known as PAMs ("protospacer adjacent motifs") serve as tags and sit adjacent to the target DNA sequence. If the Cas9 complex doesn't see a PAM next to its target DNA sequence, it won't cut. This is one possible reason thatCas9 doesn't ever attack the CRISPRregion in bacteria, according to a 2014 review published in Nature Biotechnology.

The genomes of various organisms encode a series of messages and instructions within their DNA sequences. Genome editing involves changing those sequences, thereby changing the messages. This can be done by inserting a cut or break in the DNA and tricking a cell's natural DNA repair mechanisms into introducing the changes one wants. CRISPR-Cas9 provides a means to do so.

In 2012, two pivotal research papers were published in the journalsScienceandPNAS, which helped transform bacterial CRISPR-Cas9 into a simple, programmable genome-editing tool.

The studies, conducted by separate groups, concluded that Cas9 could be directed to cut any region of DNA. This could be done by simply changing the nucleotide sequence of crRNA, which binds to a complementary DNA target. In the 2012 Science article, Martin Jinek and colleagues further simplified the system by fusing crRNA and tracrRNA to create a single "guide RNA." Thus, genome editing requires only two components: a guide RNA and the Cas9 protein.

"Operationally, you design a stretch of 20 [nucleotide] base pairs that match a gene that you want to edit," saidGeorge Church, Robert Winthrop Professor of Genetics at Harvard Medical School. An RNA molecule complementary to those 20 base pairs is constructed. Church emphasized the importance of making sure that the nucleotide sequence is found only in the target gene and nowhere else in the genome. "Then the RNA plus the protein [Cas9] will cut like a pair of scissors the DNA at that site, and ideally nowhere else," he explained.

Once the DNA is cut, the cell's natural repair mechanisms kick in and work to introduce mutations or other changes to the genome. There are two ways this can happen. According to theHuntington's Outreach Project at Stanford (University), one repair method involves gluing the two cuts back together. This method, known as "non-homologous end joining," tends to introduce errors. Nucleotides are accidentally inserted or deleted, resulting inmutations, which could disrupt a gene. In the second method, the break is fixed by filling in the gap with a sequence of nucleotides. In order to do so, the cell uses a short strand of DNA as a template. Scientists can supply the DNA template of their choosing, thereby writing-in any gene they want, or correcting a mutation.

CRISPR-Cas9 has become popular in recent years. Church notes that the technology is easy to use and is about four times more efficient than the previous best genome-editing tool (calledTALENS).

In 2013, the first reports of using CRISPR-Cas9 to edit human cells in an experimental setting were published by researchers from the laboratories ofChurchandFeng Zhangof the Broad Institute of the Massachusetts Institute of Technology and Harvard. Studies using in vitro(laboratory) and animal models of human disease have demonstrated that the technology can be effective in correcting genetic defects. Examples of such diseases includecystic fibrosis, cataracts and Fanconi anemia, according to a 2016 review article published in the journal Nature Biotechnology. These studies pave the way for therapeutic applications in humans.

CRISPR technology has also been applied in the food and agricultural industries to engineer probiotic cultures and to vaccinate industrial cultures (for yogurt, for example) against viruses. It is also being used in crops to improve yield, drought tolerance and nutritional properties.

One other potential application is to create gene drives. These are genetic systems, which increase the chances of a particular trait passing on from parent to offspring. Eventually, over the course of generations, the trait spreads through entire populations, according to theWyss Institute. Gene drives can aid in controlling the spread of diseases such as malaria by enhancing sterility among the disease vector femaleAnopheles gambiaemosquitoes according to the 2016 Nature Biotechnology article. In addition, gene drives could also be usedto eradicate invasive species and reverse pesticide and herbicide resistance,according to a 2014 article by Kenneth Oye and colleagues, published in the journal Science.

However, CRISPR-Cas9 is not without its drawbacks.

"I think the biggest limitation of CRISPR is it is not a hundred percent efficient," Church told Live Science. Moreover, the genome-editing efficiencies can vary. According to the 2014 Science article by Doudna and Charpentier, in a study conducted in rice, gene editing occurred in nearly 50 percent of the cells that received the Cas9-RNA complex. Whereas, other analyses have shown that depending on the target, editing efficiencies can reach as high as 80 percent or more.

There is also the phenomenon of "off-target effects," where DNA is cut at sites other than the intended target. This can lead to the introduction of unintended mutations. Furthermore, Church noted that even when the system cuts on target, there is a chance of not getting a precise edit. He called this "genome vandalism."

The many potential applications of CRISPR technology raise questions about the ethical merits and consequences of tampering with genomes.

In the 2014 Science article, Oye and colleagues point to the potential ecological impact of using gene drives. An introduced trait could spread beyond the target population to other organisms through crossbreeding. Gene drives could also reduce the genetic diversity of the target population.

Making genetic modifications to human embryos and reproductive cells such as sperm and eggs is known as germline editing. Since changes to these cells can be passed on to subsequent generations, using CRISPR technology to make germline edits has raised a number of ethical concerns.

Variable efficacy, off-target effects and imprecise edits all pose safety risks. In addition, there is much that is still unknown to the scientific community. In a 2015 article published in Science, David Baltimore and a group of scientists, ethicists and legal experts note thatgermline editing raises the possibility of unintended consequences for future generations"because there are limits to our knowledge of human genetics, gene-environment interactions, and the pathways of disease (including the interplay between one disease and other conditions or diseases in the same patient)."

Other ethical concerns are more nuanced. Should we make changes that could fundamentally affect future generations without having their consent? What if the use of germline editing veers from being a therapeutic tool to an enhancement tool for various human characteristics?

To address these concerns, the National Academies of Sciences, Engineering and Medicine put together acomprehensive report with guidelines and recommendationsfor genome editing.

Although the National Academies urge caution in pursuing germline editing, they emphasize "caution does not mean prohibition." They recommend that germline editing be done only on genes that lead to serious diseases and only when there are no other reasonable treatment alternatives. Among other criteria, they stress the need to have data on the health risks and benefits and the need for continuous oversight during clinical trials. They also recommend following up on families for multiple generations.

Additional resources

Broad Institute: A timeline of pivotal work on CRISPR

More here:

What Is CRISPR? - livescience.com

Researchers at the Wellcome Trust Sanger Institute and their colleagues at the University of British Columbia have developed a novel method for studying how the bacterium Chlamydia trachomatis interacts with the human immune system. Theyused a combination of gene-editing and stem cell technologies to make the model that helped lead to the discovery of two genes from our immune system, interferon regulatory factor 5 (IRF5) and interleukin-10 receptor subunit alpha (IL-10RA), as key players in fighting a Chlamydia infection.

The results, reported inNature Communications ("Exploiting Induced Pluripotent Stem Cell-Derived Macrophages to Unravel Host factor Influencing Chlamydia trachomatis Pathogenesis"), identify novel drug targets for the sexually transmitted disease.

In this study, scientists created macrophages from human induced pluripotent stem cells to study Chlamydia infection. Macrophages have a crucial role in killing Chlamydia to limit the infection. The macrophages produced responded to the disease in a similar way to those taken from human blood, meaning they are more human-like than those produced by previous methods.

This new model will enable scientists to study how Chlamydia interacts with the human immune system to avoid antibiotics and spread, according toAmy Yeung, Ph.D., first author from the Wellcome Trust Sanger Institute.

"Chlamydia is tricky to study because it can permeate and hide in macrophages where it is difficult to reach with antibiotics. Inside the macrophage, one or two Chlamydia cells can replicate into hundreds in just a day or two, before bursting out to spread the infection," she said. "This new system will allow us to understand how Chlamydia can survive and replicate in human macrophages and could have major implications for the development of new drugs."

The new model has advantages over previous methods that used macrophages either derived from mice, which differ from humans in their immune response, or immortalized human macrophage cell lines, which are genetically different from normal macrophages, she added.

In the study, scientists used CRISPR/Cas9 to genetically edit the human induced pluripotent stem cells, and then see the effects of the genetic manipulation on the resulting macrophages' ability to fight infection.

Robert Hancock, Ph.D., lead author from the University of British Columbia and associate faculty member at the Wellcome Trust Sanger Institute, noted that, "We can knock out specific genes in stem cells and look at how the gene editing influences the resulting macrophages and their interaction with Chlamydia. We're effectively sieving through the genome to find key players and can now easily see genes that weren't previously thought to be involved in fighting the infection."

The team discovered two macrophage genes in particular that were key to limiting Chlamydia infectionIRF5 and IL-10RA. When these genes were switched off, the macrophages were more susceptible to Chlamydia infection. The results suggest these genes could be drug targets for new chlamydia treatments.

"This system can be extended to study other pathogens and advance our understanding of the interactions between human hosts and infections," explainedGordon Dougan, Ph.D., senior author from the Wellcome Trust Sanger Institute. "Weare starting to unravel the role our genetics play in battling infections, such as Chlamydia, and these results could go toward designing more effective treatments in the future."

See the rest here:

CRISPR and Stem Cells Identify Novel Chlamydia Drug Targets - Genetic Engineering & Biotechnology News

Cross-Species Transplantation

One application benefiting from CRISPR/Cas9 technology is xenotransplantation, or cross-species transplantation. It offers the prospect of an unlimited supply of organs and cells, and it could resolve the critical shortage of human tissues.

For ethical and compatibility reasons, xenotransplantation shifted away from nonhuman primates as a potential source of donor tissues. Instead, the discipline began to focus on porcine organs. Nonetheless, in 1997, pig-to-human transplants were banned worldwide due to concerns about the transmission to humans of porcine endogenous retroviruses (PERVs), which are integrated into the genome of all pigs.

According to George Church, Ph.D., professor of genetics, Harvard Medical School, work was undertaken in his laboratory on PK15 porcine kidney epithelial cells to determine if PERVs could be eradicated. It was crucial to avoid disrupting the envelope gene and the terminal regulatory elements, as both of these could be important during normal pig fetal growth. In addition, a highly conserved target in the viral polymerase gene was desired for the guide RNA (gRNA) to bind.

First, the copy number of PK15 PERV was determined to be 62. Then, when CRISPR/Cas9 was used along with two gRNAs, one which did the bulk of the work, all 62 copies of the PERV pol gene were disrupted, demonstrating the possibility that PERVs could be inactivated for potential clinical pig-to-human xenotransplantation. The repeats were well separated, and not clustered, which could have meant higher toxicity.

After two weeks of cell culture, about 8% of clones were 100% altered, and no rearrangements were found. Although a few off-target effects and point mutations were expected, they were deemed unlikely to have an impact on pig fetal development. As with conventional breeding, PERV-free clones were empirically selected as they were the healthiest.

In addition to disrupting dozens of endogenous viral elements, Dr. Churchs group altered dozens of genes involved in immune and blood-clotting functions to increase human compatibility. Some of the changes were so extensive that more powerful DNA recombination tools, and not CRISPR, were utilized.

This work may benefit eGenesis, a Cambridge biotech focused on leveraging CRISPR technology to deliver safe and effective human transplantable cells, tissues, and organs. eGenesis was cofounded by Dr. Church and Luhan Yang, Ph.D., in early 2015 and is based on their research.

Continued here:

More Tooth, More Tail in CRISPR Operations | GEN - Genetic Engineering & Biotechnology News (press release)

The ability to quickly and cheaply detect minute amounts of specific nucleic acid (DNA and RNA) sequences could bring significant public health benefits. For example, it could be used to detect viral or bacterial infections in a population during outbreaks. Other possible uses include finding antibiotic-resistance genes in bacteria or tumor mutations in the body. Current methods for detecting nucleic acids involve trade-offs in sensitivity, specificity, simplicity, cost, and speed.

Drs. James J. Collins and Feng Zhang of the Broad Institute of MIT and Harvard developed a new approach by adapting the CRISPR system, which bacteria use to defend themselves from other microbes. CRISPR enzymes use short guide RNAs to identify specific target sequences to cleave. Zhangs group previously discovered that one CRISPR enzyme, called Cas13a, has an interesting collateral effect. After being activated by its target RNA sequence, Cas13a goes on to indiscriminately slice other non-targeted RNA nearby.

The researchers took advantage of this property to design a CRISPR-based nucleic acid detection platform. To detect when a target sequence was present, they added reporter RNA designed to emit a signal when cut. Whenever Cas13a was activated, it would go on to cut the reporter RNA and emit a signal. The study was funded in part by NIHs National Institute of Allergy and Infectious Diseases (NIAID) and National Institute of Mental Health (NIMH). The team described their approach online in Science on April 13, 2017.

The scientists first tested Cas13a enzymes from different bacteria to identify which had the best RNA-guided cutting activity. As amounts of DNA and RNA in samples can be minute, the researchers applied a technique called recombinase polymerase amplification, which can amplify nucleic acids without special equipment. Another enzyme could also be added to the reaction to convert DNA to RNA for Cas13a detection.

The team called this system SHERLOCK. Tests showed that SHERLOCK could detect RNA or DNA molecules at minute levels called attomolar levels. Established nucleic acid detection approaches can be similarly sensitive, but SHERLOCK gave more consistent results.

The researchers demonstrated several potential uses. SHERLOCK was able to detect specific strains of Zika and Dengue virus. It could detect Zika virus in serum, urine, and saliva from patients. It could distinguish pathogenic strains of bacteria. It could distinguish single base differences in DNA extracted from human saliva. Finally, it could detect cancer mutations among DNA fragments at levels like that found in patient blood.

Notably, SHERLOCK yielded comparable results when its components were freeze-dried, reconstituted, and tested on glass fiber paper. The scientists calculated that a paper test could be designed and created in a matter of days for as little as $0.61 per test. These qualities highlight the potential of this system for diagnostic field applications.

We can now effectively and readily make sensors for any nucleic acid, which is incredibly powerful when you think of diagnostics and research applications, Collins says. The scientific possibilities get very exciting very quickly.

This article has been republished frommaterialsprovided byNIH. Note: material may have been edited for length and content. For further information, please contact the cited source.

Read more:

Quick, Sensitive Diagnostic Tests with CRISPR - Technology Networks

DENVER--(BUSINESS WIRE)--

World licensing leader MPEG LA, LLC today invited holders of CRISPR-Cas9 patents to participate in the creation of a global CRISPR-Cas9 Joint Licensing Platform that will make their groundbreaking technologies widely accessible.

Pooling the foundational CRISPR patent rights under a single nonexclusive, cost-effective, transparent license will allow the market to focus on the creation of new products and therapies that accelerate and expand CRISPRs deployment, said Larry Horn, MPEG LA President and CEO. Just as MPEG LAs pioneering efforts to manage licensing overhead and mitigate litigation risk helped assure the success of digital video in the consumer electronics industry, the CRISPR-Cas9 Joint Licensing Platform can do the same for healthcare and other biotechnology industries but with an impact far more profound.

At the same time, both foundational and other patent owners would be rewarded for their inventions from their fair share of reasonable royalties from the pool and incentivized to develop more, added Kristin Neuman, Executive Director, Biotechnology Licensing at MPEG LA. As a voluntary market-based business solution that balances access with incentive, an independently managed pool offers the best hope for addressing market and public interests in a way that will unleash CRISPRs full potential for the benefit of humanity.

MPEG LA welcomes CRISPR-Cas9 patent holders who would like to participate in this ground floor opportunity to create a Joint Licensing Platform to visit http://www.mpegla.com/main/pid/CRISPR/default.aspx for more information, including terms and procedures governing patent submissions and eligibility. At least one eligible patent is necessary to participate in the license development process, and eligibility will be determined by MPEG LA at no cost to submitters. Interested parties are asked to make their initial patent submissions by June 30, 2017. Although submissions will continue to be accepted in order to assure that the joint license includes as much relevant intellectual property as possible for the benefit of the market, those who submit patents by that date and are found eligible will be invited to attend an initial meeting with other eligible patent rights holders to explore the potential for joint terms on which the CRISPR-Cas9 Joint Licensing Platform may be offered. Except for confidentiality, participation is without obligation or commitment, including attendance at future meetings, until such time as an eligible patent holder may decide to join the Joint Licensing Platform.

MPEG LA, LLC

MPEG LA is the worlds leading provider of one-stop licenses for standards and other technology platforms. Starting in the 1990s, it pioneered the modern-day patent pool helping to produce the most widely used standards in consumer electronics history. MPEG LA has operated licensing programs for a variety of technologies consisting of more than 14,000 patents in 84 countries with some 230 patent holders and more than 6,000 licensees. By assisting users with implementation of their technology choices, MPEG LA offers licensing solutions that provide access to fundamental intellectual property, freedom to operate, reduced litigation risk and predictability in the business planning process. In turn, this enables inventors, research institutions and other technology owners to monetize and speed market adoption of their assets to a worldwide market while substantially reducing the cost of licensing. In addition to consumer electronics, MPEG LA is developing advanced Li-Ion battery and other gene editing patent pools and has conceived licensing ventures for molecular diagnostics and oligonucleotide therapeutics. For more information, go to http://www.mpegla.com.

View source version on businesswire.com: http://www.businesswire.com/news/home/20170425005584/en/

View post:

MPEG LA Invites CRISPR-Cas9 Patents to be Pooled in a One-Stop License - Yahoo Finance

A new highly sensitive diagnostic system for diseases has been adapted from CRISPR.

Named SHERLOCK (Specific High-sensitivity Enzymatic Reporter UnLOCKing), the diagnostic systemcould detect miniscule amounts of RNA or DNA from samples and offer rapid, accurate results without the need for sophisticated lab equipment.

'This tool offers the sensitivity that could detect an extremely small amount of cancer DNA in a patient's blood sample, for example, which would help researchers understand how cancer mutates over time,' said Professor James Collins at MIT and Harvard University, who co-led the team behind the system. 'For public health, it could help researchers monitor the frequency of antibiotic-resistant bacteria in a population. The scientific possibilities get very exciting very quickly.'

The team led by Professor Collins and Professor Feng Zhang at MIT and Harvard University combined novel methods with established techniques to amplify genetic material in a sample, find a target sequence and then create a visible result.

The system uses an enzyme called Cas13a which targets RNA, and which was discovered by Professor Zhang of MIT and colleagues last year. By attaching a sequence tag, Cas13a can be guided to find and cut a specific RNA target. Once it has done this it will randomly cut any nearby pieces of 'collateral' RNA, regardless of their sequence.

The system adds so-called fluorescent reporter RNA to the samples, which emits a fluorescent signal only when cut. So if Cas13a finds its target sequence, its subsequent cutting of the reporter RNA produces a fluorescence which can be easily detected without the use of sophisticated equipment.

The precision of the enzyme is such that even a difference ofone base-pair- such as between the genetic codes of the African and American strains of Zika - will affect whether or not activation occurs. It is also able to detect concentrations as low as two molecules in a quintillion.

The system can be run in a standard test tube or on glass fibre paper, and requires no high-tech lab equipment or temperatures higher than body heat. The authors say the molecules for the test can be designed and made for as little as US $0.61.

'One thing that's especially powerful about SHERLOCK is its ability to start testing without a lot of complicated and time-consuming upstream experimental work,' said Professor Pardis Sabeti of Harvard University, a co-author of the paper. 'This ability to take raw samples and immediately start processing could transform the diagnosis of Zika and a boundless number of other infectious diseases.'

Dr Alexander McAdam, a medical microbiologist at Boston Children's Hospital who was not involved in the project, said to STAT: 'They've developed a promising method of detecting extremely low concentrations of [genetic material], but the key word is "promising".It's going to be a long walk from hopeful to clinically useful, and there is a lot to do to demonstrate practicality.'

The study was published in Science.

Excerpt from:

Highly sensitive CRISPR diagnostic tool created - BioNews