CRISPR
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Powerful Genetic Engineering Technique Could Modify Entire Wild Populations — NOVA Next | PBS

Powerful Genetic Engineering Technique Could Modify Entire Wild Populations — NOVA Next | PBS | CRISPR | Scoop.it

By combining two techniques—CRISPR and gene drives—scientists are proposing a system that could change nearly any sexually reproducing species anywhere.


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RNA-guided editing of bacterial genomes using CRISPR-Cas systems

RNA-guided editing of bacterial genomes using CRISPR-Cas systems | CRISPR | Scoop.it
A CRISPR-Cas system is harnessed to introduce template-driven mutations in S. pneumoniae and E. coli at high efficiency without requiring selectable markers.

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Gerd Moe-Behrens's curator insight, January 30, 2013 5:49 AM

Congratulation +David Bikard 

*RNA-guided editing of bacterial genomes using CRISPR-Cas systems*

by
Wenyan Jiang,David Bikard,David Cox,Feng Zhang& Luciano A Marraffini

"Here we use the clustered, regularly interspaced, short palindromic repeats (CRISPR)–associated Cas9 endonuclease complexed with dual-RNAs to introduce precise mutations in the genomes of Streptococcus pneumoniae and Escherichia coli. The approach relies on dual-RNA:Cas9-directed cleavage at the targeted genomic site to kill unmutated cells and circumvents the need for selectable markers or counter-selection systems. We reprogram dual-RNA:Cas9 specificity by changing the sequence of short CRISPR RNA (crRNA) to make single- and multinucleotide changes carried on editing templates. Simultaneous use of two crRNAs enables multiplex mutagenesis. In S. pneumoniae, nearly 100% of cells that were recovered using our approach contained the desired mutation, and in E. coli, 65% that were recovered contained the mutation, when the approach was used in combination with recombineering. We exhaustively analyze dual-RNA:Cas9 target requirements to define the range of targetable sequences and show strategies for editing sites that do not meet these requirements, suggesting the versatility of this technique for bacterial genome engineering."

http://bit.ly/11dWGDK

see also http://bit.ly/VvkdOY

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Highly efficient targeted mutagenesis in one-cell mouse embryos mediated by the TALEN and CRISPR/Cas systems - Sci. Reports

Highly efficient targeted mutagenesis in one-cell mouse embryos mediated by the TALEN and CRISPR/Cas systems - Sci. Reports | CRISPR | Scoop.it

(via T. Schreiber, thx)

Yasue et al, 2014

Since the establishment of embryonic stem (ES) cell lines, the combined use of gene targeting with homologous recombination has aided in elucidating the functions of various genes. However, the ES cell technique is inefficient and time-consuming. Recently, two new gene-targeting technologies have been developed: the transcription activator-like effector nuclease (TALEN) system, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system. In addition to aiding researchers in solving conventional problems, these technologies can be used to induce site-specific mutations in various species for which ES cells have not been established. Here, by targeting the Fgf10 gene through RNA microinjection in one-cell mouse embryos with the TALEN and CRISPR/Cas systems, we produced the known limb-defect phenotypes of Fgf10-deficient embryos at the F0 generation. Compared to the TALEN system, the CRISPR/Cas system induced the limb-defect phenotypes with a strikingly higher efficiency. Our results demonstrate that although both gene-targeting technologies are useful, the CRISPR/Cas system more effectively elicits single-step biallelic mutations in mice.


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Comparing Genome Editing Technologies

Comparing Genome Editing Technologies | CRISPR | Scoop.it
ZFN, TALEN, and CRISPR/Cas systems help scientists dissect
the vast amount of information accumulated through
the Genomic Revolution.

 

The Genomic Revolution has promised to advance medicine and biotechnology by providing scientists with enormous amounts of data that can be converted into useful information.

 

Over 10 years ago, the Human Genome Project produced the first draft of the more than 3 billion base pairs of DNA that make up the genetic code in each of our cells.

 

More recent efforts like the 1000 Genomes and HapMap Projects have since focused on identifying the differences within these billions of base pairs of DNA between individuals, while genome-wide association studies have pinpointed specific sequences that determine health and disease. The ENCODE Project and other studies have annotated chromatin states, regulatory elements, transcription factor binding sites, and other epigenetic states throughout the genome.

 

Dozens of other species have since undergone similar analyses, with the number of sequenced genomes continuously growing. Collectively, these efforts have generated an incredibly rich source of data that promises to aid our understanding of the function and evolution of any genome. However, until recently, scientists have been lacking the tools necessary to interrogate the structure and function of these elements.

 

While conventional genetic engineering methods could be used to add extra genes to cells, they cannot be easily used to modify the sequences or control the expression of genes that already exist within these genomes. These types of tools are necessary to determine not only the function of genes, but also the role of genetic variants and regulatory elements. They can also be used to overcome longstanding challenges in the field of gene therapy. Without these technologies, it has been difficult—and in some cases impossible—for scientists to capitalize on the Genomic Revolution.


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Bacterial antivirus system repurposed to attack HIV where it’s hiding

Bacterial antivirus system repurposed to attack HIV where it’s hiding | CRISPR | Scoop.it
Cuts up copies of HIV that have inserted themselves into the genome.
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Could CRISPR Transform Wild Ecosystems? - Bio-IT World

Could CRISPR Transform Wild Ecosystems? - Bio-IT World | CRISPR | Scoop.it
July 18, 2014 | In a paper published yesterday in the open access journal eLife, George Church and colleagues discuss the potential for "gene drives" that alter the genomes of whole wild populations using CRISPR gene editing technology, ...
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CRISPR Cuts Out HIV | Ultraphyte

CRISPR Cuts Out HIV | Ultraphyte | CRISPR | Scoop.it
CRISPR Cuts Out HIV. July 22, 2014. Could a bacterium's defense against bacterial viruses be used to protect a human cell from HIV? A lot is in the news about people who seemed to be “cured” of HIV (the virus causing AIDS) yet two years ...
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CRISPR and Other Genome Editing Tools Boost Medical Research ...

CRISPR and Other Genome Editing Tools Boost Medical Research ... | CRISPR | Scoop.it
Though genome editing with CRISPR is just a little over a year old, it is already reinventing genetic research. In particular, it gives scientists the ability to quickly and simultaneously make multiple genetic changes to a cell.
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CRISPR system involved in promoting antibiotic resistance - News-Medical.net

CRISPR system involved in promoting antibiotic resistance - News-Medical.net | CRISPR | Scoop.it
CRISPR system involved in promoting antibiotic resistance News-Medical.net CRISPR, a system of genes that bacteria use to fend off viruses, is involved in promoting antibiotic resistance in Francisella novicida, a close relative of the bacterium...
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Innovative Genomics Initiative launched around CRISPR-CAS9 genome editing technology

Innovative Genomics Initiative launched around CRISPR-CAS9 genome editing technology | CRISPR | Scoop.it
The University of California, Berkeley, and UC San Francisco are launching the Innovative Genomics Initiative (IGI) to lead a revolution in genetic engineering based on a new technology already generating novel strategies for gene therapy and the genetic study of disease.

 

The Li Ka Shing Foundation has provided a $10 million gift to support the initiative, establishing the Li Ka Shing Center for Genomic Engineering and an affiliated faculty chair at UC Berkeley. The two universities also will provide $2 million in start-up funds.

 

At the core of the initiative is a revolutionary technology discovered two years ago at UC Berkeley by Jennifer A. Doudna, executive director of the initiative and the new faculty chair. The technology, precision "DNA scissors" referred to as CRISPR/Cas9, has exploded in popularity since it was first published in June 2012 and is at the heart of at least three start-ups and several heavily-attended international meetings. Scientists have referred to it as the "holy grail" of genetic engineering and a "jaw-dropping" breakthrough in the fight against genetic disease. In honor of her discovery and earlier work on RNA, Doudna received last month the Lurie Prize of the Foundation for the National Institutes of Health.

 

"Professor Doudna's breakthrough discovery in genomic editing is leading us into a new era of possibilities that we could have never before imagined," said Li Ka-shing, chairman of the Li Ka Shing Foundation. "It is a great privilege for my foundation to engage with two world-class public institutions to launch the Innovative Genomics Initiative in this quest for the holy grail to fight genetic diseases."

 

In the 18 months since the discovery of this technology was announced, more than 125 papers have been published based on the technique. Worldwide, researchers are using Cas9 to investigate the genetic roots of problems as diverse as sickle cell anemia, diabetes, cystic fibrosis, AIDS and depression in hopes of finding new drug targets. Others are adapting the technology to reengineer yeast to produce biofuels and wheat to resist pests and drought.

 

The new genomic engineering technology significantly cuts down the time it takes researchers to test new therapies. CRISPR/Cas 9 allows the creation in weeks rather than years of animal strains that mimic a human disease, allowing researchers to test new therapies. The technique also makes it quick and easy to knock out genes in human cells or in animals to determine their function, which will speed the identification of new drug targets for diseases.


"We now have a very easy, very fast and very efficient technique for rewriting the genome, which allows us to do experiments that have been impossible before," said Doudna, a professor of molecular and cell biology in the California Institute for Quantitative Biosciences (QB3) and an investigator in the Howard Hughes Medical Institute at UC Berkeley. "We are grateful to Mr. Li Ka-shing for his support of our initiative, which will propel ground-breaking advances in genomic engineering."


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Crystallographic elucidation of CRISPR-Cas9 endonucleases reveal RNA-mediated conformational changes

Crystallographic elucidation of CRISPR-Cas9 endonucleases reveal RNA-mediated conformational changes | CRISPR | Scoop.it

The potential is there for bacteria and other microbes to be genetically engineered to perform a cornucopia of valuable goods and services, from the production of safer, more effective medicines and clean, green, sustainable fuels, to the clean-up and restoration of our air, water and land. Cells from eukaryotic organisms can also be modified for research or to fight disease. To achieve these and other worthy goals, the ability to precisely edit the instructions contained within a target's genome is a must. A powerful new tool for genome editing and gene regulation has emerged in the form of a family of enzymes known as Cas9, which plays a critical role in the bacterial immune system. Cas9 should become an even more valuable tool with the creation of the first detailed picture of its three-dimensional shape by researchers with the Lawrence Berkeley National Laboratory (Berkeley Lab) and the University of California (UC) Berkeley.


Biochemist Jennifer Doudna and biophysicist Eva Nogales, both of whom hold appointments with Berkeley Lab, UC Berkeley, and the Howard Hughes Medical Institute (HHMI), led an international collaboration that used x-ray crystallography to produce 2.6 and 2.2 angstrom (Å) resolution crystal structure images of two major types of Cas9 enzymes. The collaboration then used single-particle electron microscopy to reveal how Cas9 partners with its guide RNA to interact with target DNA. The results point the way to the rational design of new and improved versions of Cas9 enzymes for basic research and genetic engineering.

Cas9 is a family of RNA-guided bacterial endonucleases employed by Type II CRISPR systems to recognize and cleave double-stranded DNA at site-specific sequences. Genetic engineers have begun harnessing Cas9 for genome editing and gene regulation in many eukaryotic organisms. However, despite the successes to date, the technology has yet to reach its full potential because until now the structural basis for guide RNA recognition and DNA targeting by Cas9 has been unknown.

 

What has been a major puzzle in the CRISPR–Cas field is how Cas9 and similar RNA-guided complexes locate and recognize matching DNA targets in the context of an entire genome, the classic needle in a haystack problem," says Samuel Sternberg, lead author of the Nature paper and a member of Doudna's research group. "All of the scientists who are developing RNA-programmable Cas9 for genome engineering are relying on its ability to target unique 20-base-pair long sequences inside the cell. However, if Cas9 were to just blindly bind DNA at random sites across a genome until colliding with its target, the process would be incredibly time-consuming and probably too inefficient to be effective for bacterial immunity, or as a tool for genome engineers. Our study shows that Cas9 confines its search by first looking for PAM sequences. This accelerates the rate at which the target can be located, and minimizes the time spent interrogating non-target DNA sites."


Now, several scientists addressed this lack of detailed knowledge about Cas9 by first solving the three-dimensional crystal structures of two Cas9 proteins, representing large and small versions, from Streptococcus pyogenes (SpyCas) and Actinomyces naeslundii (AnaCas9) respectively. Using protein crystallography beamlines at Berkeley Lab's Advanced Light Source and the Paul Scherer Institute's Swiss Light Source, the collaboration discovered that despite significant differences outside of their catalytic domains, all members of the Cas9 family share the same structural core. The high resolution images showed this core to feature a clam-shaped architecture with two major lobes - a nuclease domain lobe and an alpha-helical lobe. Both lobes contained conserved clefts that become functional in nucleic acid binding.

 

"Our understanding of Cas9's structure was not complete with only the x-ray data because the protein in the crystals had been trapped in a state without its associated guide RNA," says Sam Sternberg, a member of Doudna's research group and a co-author of the Science paper. "Understanding how RNA-guided Cas9 targets matching DNA sequences for genome engineering and how this reaction and its specificity might be improved required an understanding of how the shape of Cas9 changes when it interacts with guide RNA, and when a matching DNA target sequence is bound."

 

The collaboration employed negative-staining electron microscopy to visualize the Cas9 protein bound to either guide RNA, or both RNA and target DNA. The structures revealed that the guide RNA binding structurally activates Cas9 by creating a channel between the two main lobes of the protein that functions as the DNA-binding interface.

 

"Our single particle electron microscopy analysis reveals the importance of guide-RNA for the conversion of Cas9 into a structurally-activated state," says David Taylor, a joint member of Doudna's and Nogales's research groups and another co-author of the Science paper. "The results underline that, in addition to sequence complementarity, other features of the guide-RNA must be considered when employing this technology."


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CRISPR Cas9: A novel approach to genetic engineering

Provides the basic mechanism behind the CRISPR Cas9 complex, while describing application and need.
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LABTalks: CRISPR/Cas9: Simple & versatile genome editing tool

Dr. Namritha Ravinder discusses the powerful new CRISPR/ Cas9 technology for genome editing as part of our LABTalks series.
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Application of TALEs, CRISPR/Cas and sRNAs as trans-acting regulators in prokaryotes - Curr. Opin. Biotech.

Application of TALEs, CRISPR/Cas and sRNAs as trans-acting regulators in prokaryotes - Curr. Opin. Biotech. | CRISPR | Scoop.it

(via T. Lahaye, thx)

Copeland et al, 2014

TALEs, CRISPR/Cas, and sRNAs can be easily fashioned to bind any specific sequence of DNA (TALEs, CRISPR/Cas) or RNA (sRNAs) because of the simple rules governing their interactions with nucleic acids. This unique property enables these tools to repress the expression of genes at the transcriptional or post-transcriptional levels, respectively, without prior manipulation of cis-acting and/or chromosomal target DNA sequences. These tools are now being harnessed by synthetic biologists, particularly those in the eukaryotic community, for genome-wide regulation, editing, or epigenetic studies. Here we discuss the exciting opportunities for using TALEs, CRISPR/Cas, and sRNAs as synthetic trans-acting regulators in prokaryotes.


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CRISPR technology leaps from lab to industry

CRISPR technology leaps from lab to industry | CRISPR | Scoop.it

Instead of taking prescription pills to treat their ailments, patients may one day opt for genetic 'surgery' — using an innovative gene-editing technology to snip out harmful mutations and swap in healthy DNA.
The system, called CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), has exploded in popularity in the past year, with genetic engineers, neuroscientists and even plant biologists viewing it as a highly efficient and precise research tool. Now, the gene-editing system has spun out a biotechnology company that is attracting attention from investors as well.


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GEN | Webinars:CRISPRs: Ushering in a New Age of Gene Editing

GEN | Webinars:CRISPRs: Ushering in a New Age of Gene Editing | CRISPR | Scoop.it
Get the latest in biotechnology through daily news coverage as well as analysis, features, tutorials, webinars, podcasts, and blogs. Learn about the entire bioproduct life cycle from early-stage R&D, to applied research including omics, biomarkers, as well as diagnostics, to bioprocessing and commercialization.

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Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems

Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems | CRISPR | Scoop.it

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Gerd Moe-Behrens's curator insight, March 25, 2013 7:51 PM

 by
James E. DiCarlo, Julie E. Norville, Prashant Mali, Xavier Rios, John Aach2 and George M. Church

"Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems in bacteria and archaea use RNA-guided nuclease activity to provide adaptive immunity against invading foreign nucleic acids. Here, we report the use of type II bacterial CRISPR-Cas system in Saccharomyces cerevisiae for genome engineering. The CRISPR-Cas components, Cas9 gene and a designer genome targeting CRISPR guide RNA (gRNA), show robust and specific RNA-guided endonuclease activity at targeted endogenous genomic loci in yeast. Using constitutive Cas9 expression and a transient gRNA cassette, we show that targeted double-strand breaks can increase homologous recombination rates of single- and double-stranded oligonucleotide donors by 5-fold and 130-fold, respectively. In addition, co-transformation of a gRNA plasmid and a donor DNA in cells constitutively expressing Cas9 resulted in near 100% donor DNA recombination frequency. Our approach provides foundations for a simple and powerful genome engineering tool for site-specific mutagenesis and allelic replacement in yeast."

http://bit.ly/109hfvH

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“Gene Drives” and CRISPR Could Revolutionize Ecosystem ...

“Gene Drives” and CRISPR Could Revolutionize Ecosystem ... | CRISPR | Scoop.it
The so-called “CRISPR” system naturally protects bacteria from viruses by storing fragments of viral DNA sequence and cutting any sequences that exactly match the fragment. By changing the fragments and delivering the ...
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Look Out CRISPR, BuD-Derived Gene Editing Tools May Be Gaining On You - Genetic Engineering News

Look Out CRISPR, BuD-Derived Gene Editing Tools May Be Gaining On You - Genetic Engineering News | CRISPR | Scoop.it
Genetic Engineering News
Look Out CRISPR, BuD-Derived Gene Editing Tools May Be Gaining On You
Genetic Engineering News
Look Out CRISPR, BuD-Derived Gene Editing Tools May Be Gaining On You. Kevin Mayer.
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Webinar CRISPR/Cas9 technology in haematopoietic cells and mice

Webinar CRISPR/Cas9 technology in haematopoietic cells and mice | CRISPR | Scoop.it
Abstract: The CRISPR/Cas9 technology provides an easy and rapid way to edit genes in vitro and in vivo. Initial experimental strategies utilized a transient transfection approach to modulate genes in vitro, but this proved to ...
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Bacteria Can Use CRISPR-Cas9 to Maintain Antibiotic Resistance - Genetic Engineering News

Bacteria Can Use CRISPR-Cas9 to Maintain Antibiotic Resistance - Genetic Engineering News | CRISPR | Scoop.it
Bacteria Can Use CRISPR-Cas9 to Maintain Antibiotic Resistance
Genetic Engineering News
CRISPR-Cas9, celebrated as a gene editing tool in biotechnology, has multiple functions in bacteria.
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CRISPRdirect: designing CRISPR/Cas guide RNA sequnece

http://togotv.dbcls.jp/140413.html CRISPRdirect efficiently designs guide RNA against any input DNA sequence. CRISPR/Cas system is a simple genome editing te...
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CRISPR-CAS9 Reverses Disease Symptoms in Living Animals for First Time

CRISPR-CAS9 Reverses Disease Symptoms in Living Animals for First Time | CRISPR | Scoop.it

MIT scientists report the use of a CRISPR methodology to cure mice of a rare liver disorder caused by a single genetic mutation. They say their study (“Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype”), published in Nature Biotechnology, offers the first evidence that this gene-editing technique can reverse disease symptoms in living animals. CRISPR, which provides a way to snip out mutated DNA and replace it with the correct sequence, holds potential for treating many genetic disorders, according to the research team.

 

“What's exciting about this approach is that we can actually correct a defective gene in a living adult animal,” says Daniel Anderson, Ph.D., the Samuel A. Goldblith associate professor of chemical engineering at MIT, a member of the Koch Institute for Integrative Cancer Research, and the senior author of the paper.

 

The recently developed CRISPR system relies on cellular machinery that bacteria use to defend themselves from viral infection. Researchers have copied this cellular system to create gene-editing complexes that include a DNA-cutting enzyme called Cas9 bound to a short RNA guide strand that is programmed to bind to a specific genome sequence, telling Cas9 where to make its cut.

 

At the same time, the researchers also deliver a DNA template strand. When the cell repairs the damage produced by Cas9, it copies from the template, introducing new genetic material into the genome. Scientists envision that this kind of genome editing could one day help treat diseases such as hemophilia, and others that are caused by single mutations.

 

For this study, the researchers designed three guide RNA strands that target different DNA sequences near the mutation that causes type I tyrosinemia, in a gene that codes for an enzyme called FAH. Patients with this disease, which affects about 1 in 100,000 people, cannot break down the amino acid tyrosine, which accumulates and can lead to liver failure. Current treatments include a low-protein diet and a drug called NTCB, which disrupts tyrosine production.

 

In experiments with adult mice carrying the mutated form of the FAH enzyme, the researchers delivered RNA guide strands along with the gene for Cas9 and a 199-nucleotide DNA template that includes the correct sequence of the mutated FAH gene.

 

“Delivery of components of the CRISPR-Cas9 system by hydrodynamic injection resulted in initial expression of the wild-type Fah protein in ~1/250 liver cells,” wrote the investigators. “Expansion of Fah-positive hepatocytes rescued the body weight loss phenotype.”

 

While the team used a high pressure injection to deliver the CRISPR components, Dr. Anderson envisions that better delivery approaches are possible. His lab is now working on methods that may be safer and more efficient, including targeted nanoparticles. 


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CRISPR-Cas9, Simplified.

CRISPR-Cas9, Simplified. | CRISPR | Scoop.it
[cvg-video videoId='112' width='600' height='400' mode='playlist' /]               In this short video, Mike Gibbs explains the CRISPR-Cas9 gene editing method, including a brief description of the different types of Cas enzymes.
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Quick learning of CRISPR/Cas9

What is CRISPR/Cas9? How does CRISPR system work? What is needed to perform genome engineering in your lab? This short video introduces the basics of this no...
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