Feeds:
Posts
Comments

Posts Tagged ‘CRISPR/Cas and base editing’


The Nobel Prize in Chemistry 2020: Emmanuelle Charpentier & Jennifer A. Doudna

Reporters: Stephen J. Williams, Ph.D. and Aviva Lev-Ari, PhD, RN

 

UPDATED on 11/12/2020

Harvard’s Jack Szostak congratulates former advisee Jennifer Doudna

It was a toast from one Nobel laureate to another, sweetened by the pride of a mentor to a prized student.

When Jennifer Doudna Ph.D. ’89 was honored on Wednesday with the Nobel Prize in chemistry for her work on the CRISPR gene-editing technique, she became the second person to gain such an honor from the lab of Jack Szostak, a genetics professor at Harvard Medical School and Massachusetts General Hospital, and professor of chemistry and chemical biology at Harvard’s Faculty of Arts and Sciences.

Szostak, who won the Nobel Prize in physiology or medicine in 2009 for work on how telomere caps keep the body’s chromosomes from breaking down, advised Doudna’s doctoral work on RNA and on Wednesday raised a glass in honor of Doudna, now at the University of California, Berkeley. In a tweet, Szostak expressed his delight at seeing someone he once guided through her early scientific steps soar to science’s highest reaches:

Doudna received the prize together with Emmanuelle Charpentier, for their work discovering and developing CRISPR as a precise gene-editing tool. In just the eight years since the pair announced their discovery the use of the technique has rapidly spread to a host of fields, allowing researchers to alter the code of life and develop resistant crops, new medical therapies, and even anticipate curing inherited diseases.

 

UPDADTED on 11/2/2020

 

Announcement of the Nobel Prize in Chemistry 2020

Live webcast from the press conference where the Royal Swedish Academy of Sciences will announce the Nobel Prize in Chemistry 2020.

 

 

The Royal Swedish Academy of Sciences has decided to award the Nobel Prize in Chemistry 2020 to

Emmanuelle Charpentier
Max Planck Unit for the Science of Pathogens, Berlin, Germany

Jennifer A. Doudna
University of California, Berkeley, USA

“for the development of a method for genome editing”

Genetic scissors: a tool for rewriting the code of life

Emmanuelle Charpentier and Jennifer A. Doudna have discovered one of gene technology’s sharpest tools: the CRISPR/Cas9 genetic scissors. Using these, researchers can change the DNA of animals, plants and microorganisms with extremely high precision. This technology has had a revolutionary impact on the life sciences, is contributing to new cancer therapies and may make the dream of curing inherited diseases come true.

Researchers need to modify genes in cells if they are to find out about life’s inner workings. This used to be time-consuming, difficult and sometimes impossible work. Using the CRISPR/Cas9 genetic scissors, it is now possible to change the code of life over the course of a few weeks.

“There is enormous power in this genetic tool, which affects us all. It has not only revolutionised basic science, but also resulted in innovative crops and will lead to ground-breaking new medical treatments,” says Claes Gustafsson, chair of the Nobel Committee for Chemistry.

As so often in science, the discovery of these genetic scissors was unexpected. During Emmanuelle Charpentier’s studies of Streptococcus pyogenes, one of the bacteria that cause the most harm to humanity, she discovered a previously unknown molecule, tracrRNA. Her work showed that tracrRNA is part of bacteria’s ancient immune system, CRISPR/Cas, that disarms viruses by cleaving their DNA.

Charpentier published her discovery in 2011. The same year, she initiated a collaboration with Jennifer Doudna, an experienced biochemist with vast knowledge of RNA. Together, they succeeded in recreating the bacteria’s genetic scissors in a test tube and simplifying the scissors’ molecular components so they were easier to use.

In an epoch-making experiment, they then reprogrammed the genetic scissors. In their natural form, the scissors recognise DNA from viruses, but Charpentier and Doudna proved that they could be controlled so that they can cut any DNA molecule at a predetermined site. Where the DNA is cut it is then easy to rewrite the code of life.

Since Charpentier and Doudna discovered the CRISPR/Cas9 genetic scissors in 2012 their use has exploded. This tool has contributed to many important discoveries in basic research, and plant researchers have been able to develop crops that withstand mould, pests and drought. In medicine, clinical trials of new cancer therapies are underway, and the dream of being able to cure inherited diseases is about to come true. These genetic scissors have taken the life sciences into a new epoch and, in many ways, are bringing the greatest benefit to humankind.

Illustrations

The illustrations are free to use for non-commercial purposes. Attribute ”© Johan Jarnestad/The Royal Swedish Academy of Sciences”

Illustration: Using the genetic scissors (pdf)
Illustration: Streptococcus’ natural immune system against viruses:CRISPR/Cas9 pdf)
Illustration: CRISPR/Cas9 genetic scissors (pdf)

Read more about this year’s prize

Popular information: Genetic scissors: a tool for rewriting the code of life (pdf)
Scientific Background: A tool for genome editing (pdf)

Emmanuelle Charpentier, born 1968 in Juvisy-sur-Orge, France. Ph.D. 1995 from Institut Pasteur, Paris, France. Director of the Max Planck Unit for the Science of Pathogens, Berlin, Germany.

Jennifer A. Doudna, born 1964 in Washington, D.C, USA. Ph.D. 1989 from Harvard Medical School, Boston, USA. Professor at the University of California, Berkeley, USA and Investigator, Howard Hughes Medical Institute.

SOURCE

https://www.nobelprize.org/prizes/chemistry/2020/press-release/

 

Nobel Prize in Chemistry awarded to scientists who discovered CRISPR gene editing tool for ‘rewriting the code of life’

(CNN)The Nobel Prize in Chemistry has been awarded to Emmanuelle Charpentier and Jennifer A. Doudna for the development of a method for genome editing.

They discovered one of gene technology’s sharpest tools: the CRISPR/Cas9 genetic scissors. Using these, researchers can change the DNA of animals, plants and micro-organisms with extremely high precision.
Before announcing the winners on Wednesday, Göran K. Hansson, secretary-general for the Royal Swedish Academy of Sciences, said that this year’s prize was about “rewriting the code of life.”
The American biochemist Jennifer A. Doudna (left) and French microbiologist Emmanuelle Charpentier, pictured together in 2016.
 
The CRISPR/Cas9 gene editing tools have revolutionized the molecular life sciences, brought new opportunities for plant breeding, are contributing to innovative cancer therapies and may make the dream of curing inherited diseases come true, according to a press release from the Nobel committee.
 
 
There have also been some ethical concerns around the CRISPR technology, however.
Charpentier, a French microbiologist, and Doudna, an American biochemist, are the first women to jointly win the Nobel Prize in Chemistry, and the sixth and seventh women to win the chemistry prize.
close dialog

 

Jennifer Doudna wins 2020 Nobel Prize in chemistry

 

First Day in a Nobel Life: Jennifer Doudna

12,365 views
Oct 7, 2020
 
Scenes from day that UC Berkeley Professor Jennifer Doudna won the Nobel Prize For the full story, visit: https://news.berkeley.edu/2020/10/07/… University of California, Berkeley, biochemist Jennifer Doudna today won the 2020 Nobel Prize in Chemistry, sharing it with colleague Emmanuelle Charpentier for the co-development of CRISPR-Cas9, a genome editing breakthrough that has revolutionized biomedicine. CRISPR-Cas9 allows scientists to rewrite DNA — the code of life — in any organism, including human cells, with unprecedented efficiency and precision. The groundbreaking power and versatility of CRISPR-Cas9 has opened up new and wide-ranging possibilities across biology, agriculture and medicine, including the treatment of thousands of intractable diseases. Doudna and Charpentier, director of the Max Planck Institute for Infection Biology, will share the 10 million Swedish krona (more than $1 million) prize. “This great honor recognizes the history of CRISPR and the collaborative story of harnessing it into a profoundly powerful engineering technology that gives new hope and possibility to our society,” said Doudna. “What started as a curiosity‐driven, fundamental discovery project has now become the breakthrough strategy used by countless researchers working to help improve the human condition. I encourage continued support of fundamental science as well as public discourse about the ethical uses and responsible regulation of CRISPR technology.” Video by Clare Major & Roxanne Makasdjian
SOURCE

 

Jennifer Doudna wins 2020 Nobel Prize in chemistry

 

Jennifer Doudna in the PBS Movie CRISPR

Our critically-acclaimed documentary HUMAN NATURE is now streaming on NETFLIX. #HumanNatureFilm. Find out more about the film on our website.

 

Other Articles on the Nobel Prize in this Open Access Journal Include:

2020 Nobel Prize for Physiology and Medicine for Hepatitis C Discovery goes to British scientist Michael Houghton and US researchers Harvey Alter and Charles Rice

CONTAGIOUS – About Viruses, Pandemics and Nobel Prizes at the Nobel Prize Museum, Stockholm, Sweden 

AACR Congratulates Dr. William G. Kaelin Jr., Sir Peter J. Ratcliffe, and Dr. Gregg L. Semenza on 2019 Nobel Prize in Physiology or Medicine

2018 Nobel Prize in Physiology or Medicine for contributions to Cancer Immunotherapy to James P. Allison, Ph.D., of the University of Texas, M.D. Anderson Cancer Center, Houston, Texas. Dr. Allison shares the prize with Tasuku Honjo, M.D., Ph.D., of Kyoto University Institute, Japan

2017 Nobel prize in chemistry given to Jacques Dubochet, Joachim Frank, and Richard Henderson  for developing cryo-electron microscopy

2016 Nobel Prize in Chemistry awarded for development of molecular machines, the world’s smallest mechanical devices, the winners: Jean-Pierre Sauvage, J. Fraser Stoddart and Bernard L. Feringa

Correspondence on Leadership in Genomics and other Gene Curations: Dr. Williams with Dr. Lev-Ari

Programming life: An interview with Jennifer Doudna by Michael Chui, a partner of the McKinsey Global Institute

Read Full Post »


Recent Progress in Gene Editing Error Reduction

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Advances in Genome Editing

Researchers develop a CRISPR-based technique that efficiently corrects point mutations without cleaving DNA.

By Catherine Offord | April 20, 2016

http://www.the-scientist.com/?articles.view/articleNo/45903/title/Advances-in-Genome-Editing

http://www.the-scientist.com/images/News/April2016/dnaligase2.jpg

Illustration of DNA ligase, one of the cell proteins involved in repairing double-strand breaks in DNA WIKIMEDIA; WASHINGTON UNIVERSITY SCHOOL OF MEDICINE IN ST. LOUIS, TOM ELLENBERGER

Most genetic diseases in humans are caused by point mutations—single base errors in the DNA sequence. However, current genome-editing methods cannot efficiently correct these mutations in cells, and often cause random nucleotide insertions or deletions (indels) as a byproduct. Now, researchers at Harvard University have modified CRISPR/Cas9 technology to get around these problems, creating a new “base editor,” described today (April 20) in Nature, which permanently and efficiently converts cytosine (C) to uracil (U) bases with low error in human and mouse cell lines.

“There are a lot of genetic diseases where you would want, in essence, to swap bases in and out,” said Jacob Corn, scientific director of the Innovative Genomics Initiative at the University of California, Berkeley, who was not involved in the research. “Trying to get this to work is one of the big challenges in the field, and I think this is a really exciting approach.”

To date, CRISPR/Cas9 genome-editing approaches have relied on a cellular mechanism called homology-directed repair, which is triggered by double-strand breaks in DNA. Researchers supply cells with a template containing the desired sequence, make a targeted double-strand break with the Cas9 enzyme, and then wait to see whether homology-directed repair incorporates the template to reconnect the strands. Unfortunately, this method is inefficient (incorporation is rare) and often introduces new errors in the form of random indels around the break, making it impractical for therapeutic correction of point mutations.

So researchers at Harvard, led by chemist and chemical biologist David Liu, tried a different approach. First, they inactivated part of Cas9 so that it couldn’t make the double-strand break. They then tethered Cas9 to an enzyme called cytidine deaminase that directly catalyzes conversion of C to U (essentially an equivalent of thymine, T), without DNA cleavage. Sending this machinery into cells creates a mismatched pair at the target, comprising the newly introduced U, and an original guanine base (G) on the opposite strand. “This [mismatch] distorts the DNA,” Liu explained. “It creates a funny little bulge that doesn’t look normal.”

The bulge alerts a different cellular repair mechanism, mismatch repair, which removes one of the mismatched bases and replaces it with the complement to the remaining one. Without any information about which base is incorrect, mismatch repair produces the desired G to A conversion about 50 percent of the time; the rest of the time it converts the U back into a C.

But mismatch repair does incorporate further information when available: it detects tiny breaks in the DNA backbone called nicks. “Cells have evolved mismatch repair machinery to prioritize old DNA over newly synthesized DNA,” said Liu. “Newly synthesized DNA tends to have some nicks in it. So we reasoned that we could manipulate mismatch repair to favor correcting the DNA strand that we don’t want, namely the strand containing the G.”

The team again modified Cas9, this time so that it would create a nick in the nonedited, G-containing strand, while leaving the edited, U-containing strand intact. “Now the cell says, ‘Aha, there’s a mismatch here, and the base at fault must be the G, because that must be a newly synthesized strand because it has a nick in it,’” said Liu. “It will preferentially correct that G, using the other strand as a template.”

Using the technique at six loci in human cells, the team reported a targeted base correction rate of up to 37 percent, with only around 1 percent of the sequences showing indels. By contrast, a normal Cas9 editing technique tested on three of those loci showed less than one percent efficiency, and more than four percent formation of indels. The researchers also demonstrated the technique’s potential to correct disease-associated mutations by converting a variant of APOE, a gene linked with Alzheimer’s, into a lower risk version in mouse cells.

“By engineering this Cas9, they’ve figured out a really nice way to trick the cell into preferring pathways that it would normally not prefer,” said Corn. However, because the method is currently only able to convert C-G to U-A (i.e., T-A) base pairs, and in some cases edits other C bases in the immediate vicinity of the target, “it’s certainly not a panacea,” he cautioned. “It doesn’t mean that you can now cure every genetic disease out there. But there are probably going to be quite a few that fit into this category.”

The University of Oxford’s Tudor Fulga called the technique “an extremely ingenious idea” to get around inefficient homology-directed repair, and to reduce unwanted indel formation. “I think this will set up a paradigm shift in the field,” he told The Scientist. “It is very likely that the impact of Cas9-mediated base editing is going to be massive—both in terms of answering basic research questions and in genome engineering–based therapeutic applications.”

Also appearing in Nature today are two studies addressing a potential alternative to Cas9: the Cpf1 enzyme. CRISPR/Cpf1 creates “sticky ends”—overhangs in cleaved DNA that leave unpaired bases either side of the break—rather than the blunt ends made by Cas9’s double-strand DNA cleavage.

Emmanuelle Charpentier and colleagues at the Max Planck Institute for Infection Biology in Germany have shown that, unlike Cas9, Cpf1 processes RNA in addition to cleaving DNA. Meanwhile, Zhiwei Huang of the Harbin Institute of Technology, China, and colleagues have described the crystal structure of CRISPR/Cpf1.

“Sticky ends are more efficient [than blunt ends] for DNA repair in cells,” Huang told The Scientist. “We believe that [understanding] the structure of Cpf1 will help us not only to know the working mechanism of Cpf1 but also to design more specific and more efficient genome-editing tools.”

D. Dong et al., “The crystal structure of Cpf1 in complex with CRISPR RNA,” Nature, doi:10.1038/nature17944, 2016.

I. Fonfara et al., “The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA,” Nature, doi:10.1038/nature17945, 2016.

A.C. Komor et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage,” Nature, doi:10.1038/nature17946, 2016.

 

Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage

Alexis C. KomorYongjoo B. KimMichael S. PackerJohn A. Zuris & David R. Liu
Nature(2016)
       doi:10.1038/nature17946

Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction1, 2. Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus resulting from the cellular response to dsDNA breaks1, 2. Here we report the development of ‘base editing’, a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting ‘base editors’ convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor, and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favour desired base-editing outcomes, resulting in permanent correction of ~15–75% of total cellular DNA with minimal (typically ≤1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.

http://www.nature.com/nature/journal/vaop/ncurrent/fig_tab/nature17946_F1.html

http://www.nature.com/nature/journal/vaop/ncurrent/carousel/nature17946-f2.jpg

http://www.nature.com/nature/journal/vaop/ncurrent/carousel/nature17946-f3.jpg

 

Alternative CRISPR system could improve genome editing

Smaller enzyme may make process simpler and more exact.

http://www.nature.com/news/alternative-crispr-system-could-improve-genome-editing-1.18432

The CRISPR/Cas9 technique is revolutionizing genetic research: scientists have already used it to engineer crops, livestock and even human embryos, and it may one day yield new ways to treat disease.

But now one of the technique’s pioneers thinks that he has found a way to make CRISPR even simpler and more precise. In a paper published in Cell on 25 September, a team led by synthetic biologist Feng Zhang of the Broad Institute in Cambridge, Massachusetts, reports the discovery of a protein1 called Cpf1 that may overcome one of CRISPR/Cas9’s few limitations; although the system works well for disabling genes, it is often difficult to truly edit them by replacing one DNA sequence with another.

The CRISPR/Cas9 system evolved as a way for bacteria and archaea to defend themselves against invading viruses. It is found in a wide range of these organisms, and uses an enzyme called Cas9 to cut DNA at a site specified by ‘guide’ strands of RNA. Researchers have turned CRISPR/Cas9 into a molecular-biology powerhouse that can be used in other organisms. The cuts made by the enzyme are repaired by the cell’s natural DNA-repair processes.

Good, better, best?

CRISPR is much simpler than previous gene-editing methods, but Zhang thought there was still room for improvement.

So he and his colleagues searched the bacterial kingdom to find an alternative to the Cas9 enzyme commonly used in laboratories. In April, they reported that they had discovered a smaller version of Cas9 in the bacterium Staphylococcus aureus2. The small size makes the enzyme easier to shuttle into mature cells — a crucial destination for some potential therapies.

The team was also intrigued by Cpf1, a protein that looks very different from Cas9, but is present in some bacteria with CRISPR. The scientists evaluated Cpf1 enzymes from 16 different bacteria, eventually finding two that could cut human DNA.

They also uncovered some curious differences between how Cpf1 and Cas9 work. Cas9 requires two RNA molecules to cut DNA; Cpf1 needs only one. The proteins also cut DNA at different places, offering researchers more options when selecting a site to edit. “This opens up a lot of possibilities for all the things we could not target before,” says epigeneticist Luca Magnani of Imperial College London.

Cpf1 also cuts DNA in a different way. Cas9 cuts both strands in a DNA molecule at the same position, leaving behind what molecular biologists call ‘blunt’ ends. But Cpf1 leaves one strand longer than the other, creating a ‘sticky’ end. Blunt ends are not as easy to work with: a DNA sequence could be inserted in either end, for example, whereas a sticky end will only pair with a complementary sticky end.

“The sticky ends carry information that can direct the insertion of the DNA,” says Zhang. “It makes the insertion much more controllable.”

Zhang’s team is now working to use these sticky ends to improve the frequency with which researchers can replace a natural DNA sequence. Cuts left by Cas9 tend to be repaired by sticking the two ends back together, in a relatively sloppy repair process that can leave errors. Although it is possible that the cell will instead insert a designated, new sequence at that site, that kind of repair occurs at a much lower frequency. Zhang hopes that the unique properties of how Cpf1 cuts may be harnessed to make such insertions more frequent.

For Bing Yang, a plant biologist at the Iowa State University in Ames, this is the most exciting aspect of Cpf1. “Boosting the efficiency would be a big step for plant science,” he says. “Right now, it is a major challenge.”

Will the new enzyme surpass Cas9 in popularity? “It’s too early to tell,” says Zhang. “It certainly has some distinct advantages.” The CRISPR/Cas9 system is so popular — and potentially lucrative — that it has sparked a fierce patent fight between the University of California, Berkeley, and the Broad Institute and its ally, the Massachusetts Institute of Technology in Cambridge. Zhang says that his lab will make the CRISPR/Cpf1 components available to academic researchers, as it has done with its CRISPR/Cas9 tools.

For now, the results stand as a testament that researchers still have more to learn from the genome-editing systems that bacteria have evolved. “This study powerfully demonstrates that the natural evolutionary diversity of CRISPR systems is rich with potential solutions to the challenges facing the use of genome-editing agents,” says David Liu, a chemical biologist at Harvard University in Cambridge. (Zhang and Liu are both scientific advisers to Editas Medicine, a company in Cambridge that aims to develop CRISPR-based therapies.)

Microbiologist John van der Oost of Wageningen University in the Netherlands, who collaborated on the latest study with Zhang, plans to keep searching for new methods. “You never know whether one of these systems will be suitable for genome editing,” he says. “There are still surprises ahead of us.”

Nature 526, 17 (01 October 2015)    http://dx.doi.org:/10.1038/nature.2015.18432
  1. Zetsche, B. et al. Cell http://dx.doi.org/10.1016/j.cell.2015.09.038 (2015).
  2. Ran, F. A. et al. Nature 520, 186191 (2015).

CRISPR 2.0?

A pioneer of the gene-editing technique discovers a protein that could improve its accuracy.

By Jef Akst | September 28, 2015     http://www.the-scientist.com/?articles.view/articleNo/44115/title/CRISPR-2-0-/

Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System 

Bernd Zetsche10, Jonathan S. Gootenberg10, Omar O. Abudayyeh,…, Aviv Regev, Eugene V. Koonin, Feng Zhang10Co-first author
  • CRISPR-Cpf1 is a class 2 CRISPR system
  • Cpf1 is a CRISPR-associated two-component RNA-programmable DNA nuclease
  • Targeted DNA is cleaved as a 5-nt staggered cut distal to a 5′ T-rich PAM
  • Two Cpf1 orthologs exhibit robust nuclease activity in human cells

The microbial adaptive immune system CRISPR mediates defense against foreign genetic elements through two classes of RNA-guided nuclease effectors. Class 1 effectors utilize multi-protein complexes, whereas class 2 effectors rely on single-component effector proteins such as the well-characterized Cas9. Here, we report characterization of Cpf1, a putative class 2 CRISPR effector. We demonstrate that Cpf1 mediates robust DNA interference with features distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif. Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpf1-family proteins, we identified two candidate enzymes from Acidaminococcus and Lachnospiraceae, with efficient genome-editing activity in human cells. Identifying this mechanism of interference broadens our understanding of CRISPR-Cas systems and advances their genome editing applications.

http://www.cell.com/cms/attachment/2040452728/2053961450/fx1.jpg

 

The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA

Ines FonfaraHagen RichterMajda BratovičAnaïs Le Rhun & Emmanuelle Charpentier
Nature(2016)doi:10.1038/nature17945

CRISPR–Cas systems that provide defence against mobile genetic elements in bacteria and archaea have evolved a variety of mechanisms to target and cleave RNA or DNA1. The well-studied types I, II and III utilize a set of distinct CRISPR-associated (Cas) proteins for production of mature CRISPR RNAs (crRNAs) and interference with invading nucleic acids. In types I and III, Cas6 or Cas5d cleaves precursor crRNA (pre-crRNA)2, 3, 4, 5 and the mature crRNAs then guide a complex of Cas proteins (Cascade-Cas3, type I; Csm or Cmr, type III) to target and cleave invading DNA or RNA6, 7, 8, 9, 10, 11, 12. In type II systems, RNase III cleaves pre-crRNA base-paired withtrans-activating crRNA (tracrRNA) in the presence of Cas9 (refs 13, 14). The mature tracrRNA–crRNA duplex then guides Cas9 to cleave target DNA15. Here, we demonstrate a novel mechanism in CRISPR–Cas immunity. We show that type V-A Cpf1 from Francisella novicida is a dual-nuclease that is specific to crRNA biogenesis and target DNA interference. Cpf1 cleaves pre-crRNA upstream of a hairpin structure formed within the CRISPR repeats and thereby generates intermediate crRNAs that are processed further, leading to mature crRNAs. After recognition of a 5′-YTN-3′ protospacer adjacent motif on the non-target DNA strand and subsequent probing for an eight-nucleotide seed sequence, Cpf1, guided by the single mature repeat-spacer crRNA, introduces double-stranded breaks in the target DNA to generate a 5′ overhang16. The RNase and DNase activities of Cpf1 require sequence- and structure-specific binding to the hairpin of crRNA repeats. Cpf1 uses distinct active domains for both nuclease reactions and cleaves nucleic acids in the presence of magnesium or calcium. This study uncovers a new family of enzymes with specific dual endoribonuclease and endonuclease activities, and demonstrates that type V-A constitutes the most minimalistic of the CRISPR–Cas systems so far described.

 

The crystal structure of Cpf1 in complex with CRISPR RNA

Nature(2016)    http://dx.doi.org:/10.1038/nature17944http://dx.doi.org:/10.1038/nature17944

The CRISPR–Cas systems, as exemplified by CRISPR–Cas9, are RNA-guided adaptive immune systems used by bacteria and archaea to defend against viral infection1, 2, 3, 4, 5, 6, 7. The CRISPR–Cpf1 system, a new class 2 CRISPR–Cas system, mediates robust DNA interference in human cells1, 8, 9, 10. Although functionally conserved, Cpf1 and Cas9 differ in many aspects including their guide RNAs and substrate specificity. Here we report the 2.38 Å crystal structure of the CRISPR RNA (crRNA)-bound Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1). LbCpf1 has a triangle-shaped architecture with a large positively charged channel at the centre. Recognized by the oligonucleotide-binding domain of LbCpf1, the crRNA adopts a highly distorted conformation stabilized by extensive intramolecular interactions and the (Mg(H2O)6)2+ ion. The oligonucleotide-binding domain also harbours a looped-out helical domain that is important for LbCpf1 substrate binding. Binding of crRNA or crRNA lacking the guide sequence induces marked conformational changes but no oligomerization of LbCpf1. Our study reveals the crRNA recognition mechanism and provides insight into crRNA-guided substrate binding of LbCpf1, establishing a framework for engineering LbCpf1 to improve its efficiency and specificity for genome editing

 

Read Full Post »