The CRISPR/Cas system is an immune system currently found in most bacteria and all archaea and is used to identify and destroy defense systems against phage and other pathogens. In the CRISPR/Cas system, CRISPR is an abbreviation for clustered regular interspaced short palindromic repeats, which involves a unique DNA region in the bacterial genome and also stores viral DNA fragments to allow cells to recognize any Where an attempt is made to re-infect its virus, CRISPR transcribed RNA sequences (known as crRNA) recognize the genetic material of the invading virus. Cas is short for CRISPR-associated proteins (Cas), which cleaves target DNA on the bacterial genome like a molecular scissors.
The survival battle between bacteria and bacteria-infected viruses (phage) has led to the evolution of many bacterial defense systems, and phage has evolved new antagonists against these systems: anti-CRISPR proteins. The CRISPR-Cas system is one of the most common methods by which bacteria protect themselves against phage, and it plays an important role as an adaptive immune system in many bacteria. At present, the CRISPR-Cas9 system has been widely used by scientists for genetic modification and gene function research.
Given that it is known that Cas9 endonuclease stays in the cell for too long to cause off-target effects, scientists are working hard to develop a Cas9 shutdown switch. Ideally, Cas9 will find a perfect target, followed by a Cas9 inhibitor to block the enzyme. Additional cutting occurs. Now studies have confirmed that the virus's evolutionary counter-measure, called the anti-CRISPR protein (anti-CRISPR) inhibitory protein, can be used to improve CRISPR-Cas9 as a gene therapy tool, thereby reducing the possible Off-target gene editing for side effects.
Prior to this, Xiaobian conducted a large number of summarization and analysis on the CRISPR/Cas system, but did not specifically summarize the anti-CRISPR protein. Based on this, Xiao Bian specially conducted an inventory of anti-CRISPR protein progress in recent years to readers.
1.Nature: CRISPR-Cas also has natural enemies!
Doi:10.1038/nature15254
Recently, researchers from the University of Toronto in Canada published a new research progress in the famous international academic journal Nature. In this study, they first discovered the protein synthesized by phage to inhibit the CRISPR-CAS system in bacteria. In this study, the researchers used biochemical and in vivo studies to study the three anti-CRISPR proteins produced by phage, and found that each protein inhibits CRISPR-CAS activity through different mechanisms. Two of these proteins block the DNA-binding activity of the CRISPR-CAS complex by interacting with different protein subunits in the CRISPR-CAS complex, using steric or non-spatial inhibitory effects. The third anti-CRISPR protein, by binding to the CAS3 helicase-nuclease, prevents it from being recruited to the DNA-binding CRISPR-CAS complex.
Using in vivo experiments, the researchers demonstrated that anti-CRISPR can transform the CRISPR-CAS system into a transcriptional repressor, and for the first time proposed a model that regulates CRISPR-CAS activity through protein binding.
The sequence and mechanism of action of these anti-CRISPR proteins represent their independent evolutionary processes, suggesting that there may be other proteins that may regulate CRISPR-CAS function, and more research is needed for further investigation.
2. Cell: Like! Also discovered two new anti- CRISPR proteins
Doi:10.1016/j.cell.2016.12.009
Just after discovering several proteins that block CRISPR-Cas9 activity in human cells (Cell, doi:10.1016/j.cell.2016.11.017), Joseph Bondy-Denomy and colleagues from the University of California, San Francisco are More anti-CRISPR proteins (anti-CRISPR) have been reported in a new study.
The researchers first hypothesized that the bacterial genome might contain an inhibitor to prevent CRISPR from cleavage of its own target in the genome, and then discover these Cas9 inhibitors by looking for the CRISPR sequence and its target in the bacterial genome. Indeed, Rauch and colleagues revealed several anti-CRISPR proteins present in Listeria, and the sequences of these anti-CRISPR proteins were retained in the Listeria genome in previous phage infections. Rachel said, "As CRISPR technology is based on a natural antiviral defense system based on bacteria, we can also use the anti-CRISPR protein produced by the virus to bypass these bacterial defense systems."
The study found that two protein inhibitors, AcrIIA2 and AcrIIA4, block Cas9 enzymatic activity from S. pyogenes, a DNA cleavage enzyme often used in genome editing.
3. Nat Microbiol: Microorganisms use the anti-CRISPR protein to destroy the CRISPR/Cas system
Doi:10.1038/nmicrobiol.2016.85
In a new study, a team from the University of Toronto in Canada and the University of Otago in New Zealand found that diverse bacterial species encode genes that block specific CRISPR/Cas system activity. By scanning the prophage (ie, the phage genome integrated into the bacterial genome), Alan Davidson and colleagues from the University of Toronto identified five new anti-CRISPR protein-encoding genes, and his team has identified nine previously. . This latest study highlights a way to learn how the CRISPR system works and a potential additional tool for developing CRISPR-based gene editing. The relevant research results were published online June 13, 2016 in the journal Nature Microbiology, entitled "Inactivation of CRISPR-Cas systems by anti-CRISPR proteins in diverse bacterial species".
In the current study, the Davidson team expanded the search for the genome of bacterial species in Proteobacteria. Given that the nine proteins previously identified do not share a common sequence motif, the researchers looked for sequences that are homologous to a putative transcriptional regulatory gene because they found that this transcriptional regulatory gene is located in all known resistances. Near the CRISPR site.
Davidson and colleagues then tested whether each putative anti-CRISPR gene expressed in the plasmid was able to support bacteriophage replication in bacteria carrying the IE-type or IF-type CRISPR system targeting it. They also identified four anti-CRISPR genes that target the IF-type CRISPR system, as well as an anti-CRISPR gene that blocks the IE- and IF-type CRISPR systems.
4.mBio: Identification of a new set of phage anti-CRISPR genes that inhibit the Pseudomonas aeruginosa IE-type CRISPR-Cas system
Doi:10.1128/mBio.00896-14
In a new study, the Alan Davidson team at the University of Toronto confirmed that some phage carrying the IF anti-CRISPR gene mediate inhibition of the IE-type CRISPR-Cas system of P. aeruginosa. These genes do not inhibit the IF-type CRISPR-Cas system of P. aeruginosa, nor do they inhibit the IE-type CRISPR-Cas system of E. coli. They are also in the non-prophage genomic region of P. aeruginosa (possibly mobile) A functional anti-CRISPR gene was identified in the genetic factors).
They confirmed for the first time that Pseudomonas aeruginosa's IE-type CRISPR-Cas system is naturally active without genetic manipulation, in stark contrast to the previously described E. coli and other bacterial IE-type CRISPR-Cas systems.
5.Cell: Heavy! Revealing the mechanism of anti-CRISPR protein blocking CRISPR system
Doi:10.1016/j.cell.2017.03.012
Now, researchers from the National Institute of Allergy and Infectious Diseases, Scripps Research Institute, Montana State University, University of California, San Francisco, and the University of Toronto, Canada, have first analyzed the adhesion of viral anti-CRISPR proteins to a bacterial CRISPR. The structure when monitoring the composite. They found that the mechanism of action of anti-CRISPR proteins is to block the ability of CRISPR to recognize and attack the viral genome. An anti-CRISPR protein or even "mock" DNA that decouples this crRNA (CRISPR-transcribed RNA)-guided detection machine. The results of the study were published in the March 23, 2017 issue of Cell, entitled "Structure Reveals Mechanisms of Viral Suppressors that Intercept a CRISPR RNA-Guided Surveillance Complex." The authors of the paper were Gabriel C. Lander from the Scripps Research Institute and Blake Wiedenheft from Montana State University.
Using a high-resolution imaging technique called cryo-EM, these researchers discovered three important aspects of the CRISPR system and anti-CRISPR proteins.
First, they accurately observed how this CRISPR surveillance complex recognizes viral genetic material in order to find out where it should launch an attack. The protein in this surveillance complex wraps around the bacterial crRNA like a handshake, exposing specific fragments of this crRNA. This particular fragment scans the viral DNA for the sequence of genes it can recognize. Furthermore, these researchers analyzed how viral anti-CRISPR proteins lick such surveillance complexes. They found that an anti-CRISPR protein covers this exposed fragment of crRNA, preventing the CRISPR system from scanning viral DNA.
Another anti-CRISPR protein uses a different strategy. Based on the binding position and negative charge of this anti-CRISPR protein, these researchers believe that it acts as a mimicking DNA that induces CRISPR binding to this anti-CRISPR protein rather than invading viral DNA.
These researchers believe that this new understanding of CRISPR proteins may eventually lead to the development of more complex and more efficient gene editing tools. Anti-CRISPR proteins may be used in the CRISPR system to rapidly block gene editing, or scientists may be able to degrade anti-CRISPR proteins to trigger gene editing.
6.Science Supplement: Using anti-CRISPR protein to significantly reduce the off-target effect of CRISPR-Cas9
Doi:10.1126/sciadv.1701620
Now studies have confirmed that the virus's evolutionary counter-measure, called the anti-CRISPR protein (anti-CRISPR) inhibitory protein, can be used to improve CRISPR-Cas9 as a gene therapy tool, thereby reducing the possible Off-target gene editing for side effects.
In a new study, researchers from the University of California at Berkeley and the University of California at San Francisco confirmed that the recently discovered anti-CRISPR protein reduced the off-target effect by up to four times, allowing the CRISPR-Cas9 to complete it like a cut-off switch. Lost function after the task. The results of the study were published in the July 12, 2017 issue of Science Advances, entitled "Disabling Cas9 by an anti-CRISPR DNA mimic." The author of the paper is Dr. Jacob Corn, a researcher at the Institute of Innovative Genomics at the University of California at Berkeley, and Jennifer A. Doudna, one of the inventors of the CRISPR-Cas9 gene editor from the University of California at Berkeley.
In this study, the researchers asked the CRISPR-Cas9 molecule to use the guide RNA (gRNA) to discover, cleave, and replace hemoglobin-encoding gene mutations that cause sickle cell disease. They demonstrated that a specific anti-CRISPR protein called AcrIIA4 reduced the off-target effect of this CRISPR-Cas9 molecule by a factor of four. It does not significantly reduce the desired target gene editing while doing this.
7.Nature Supplement: Zhu Yongqun Laboratory of Zhejiang University Reveals the Molecular Mechanism of AcrF3 Protein Inhibiting the Immune System of Pathogen Type I CRISPR-Cas
Doi:10.1038/nsmb.3269
Schematic diagram of crystal structure of AcrF3-PaCas3 complex
On July 25, 2016, Zhu Yongqun Laboratory of Zhejiang University Life Science Research Institute was published online in the journal Nature Structural & Molecular Biology, a publication of the International Academic Authoritative Publications Nature Publishing Group. “Structural Basis for Cas3 Inhibition by the Bacteriophage Protein AcrF3" research paper reveals that the phage protein AcrF3 inhibits the molecular mechanism of the pathogen type I CRISPR-Cas immune system effector molecule Cas3. Dr. Yao Deqiang from the National Protein Science Center and Dr. Wang Xiaofei from the laboratory of Zhu Yongqun are the co-first authors of the paper. Professor Zhu Yongqun and assistant researcher Dr. Zhou Yan are the co-authors of this article.
Zhu Yongqun laboratory established the in vitro nuclease enzymatic experimental system of PaCas3, and found that PaCas3 has a good variety of divalent metal ion-dependent nuclease activities. Further biochemical experiments revealed that the AcrF3 protein forms a homodimeric molecule in solution and specifically binds to PaCas3 in a high affinity manner, effectively inhibiting the in vitro nuclease activity of PaCas3. By analyzing the crystal structure of the 2.6 angstrom high-resolution PaCas3-AcrF3 complex, the study found that PaCas3 consists of a separate N-terminal Cas2 domain, HD-type nuclease domain, and SF2-type helicase domain (by RecA1 and RecA2). It consists of two subdomains, a Long linker region, and a C-terminal domain (CTD). The Cas2 protein alone is reported to play a key role in the DNA adaptation process of the CRISPR-Cas immune system. Cas2 forms a 2:4 integrase complex with Cas1, and the acquired foreign DNA is inserted into the CRISPR locus. in. PaCas3 has an independent Cas2 domain, indicating that PaCas3 is different from the previously reported Cas3 molecule, and it is involved in both DNA adaptation and DNA interference in the CRISPR-Cas system.
In the crystal structure of PaCas3-AcrF3 complex, AcrF3 is tightly bound to PaCas3 by homodimer, which is completely consistent with our biochemical experiments. A single AcrF3 molecule exhibits a bilayer structure consisting of six alpha-helices and forms a homodimeric molecule in a reverse symmetric manner by hydrophobic interaction. Destruction of the hydrophobic interaction between the two molecules in the dimer will result in the complete loss of AcrF3's ability to inhibit the in vitro nuclease activity of PaCas3. In the complex structure, the AcrF3 dimer binds to the groove region between the Long linker region and the CTD domain of PaCas3, and occurs extensively with the HD, Long linker region, RecA2 and CTD domains of PaCas3. interaction. Among them, the interaction between AcrF3 and RecA2 completely blocked the entry of the DNA binding channel in the PaCas3 helicase domain, indicating that the binding of AcrF3 resulted in PaCas3 not being able to contact the target DNA presented by Cascade. Surprisingly, in the complex structure, the PaCas3 helicase domain binds to an ADP molecule rather than the ATP molecule that normally binds to the active helicase, indicating that AcrF3 locks PaCas3 in an inactive state. Through further structural analysis, the study also found that AcrF3 also prevented the recognition of PaCas3 by the Cse1 subunit of the Cascade complex, resulting in Cascade not recruiting PaCas3. Therefore, the AcrF3 protein acts as a living unit with homodimers, inhibits the contact of PaCas3 with the target DNA, blocks the recruitment of the Cascade complex Cse1 subunit to PaCas3, and locks PaCas3 in the inactive state of ADP binding. The type I CRISPR-Cas immune system exerts its anti-CRISPR activity.
8.Nature: discovery of phage genes that inactivate the CRISPR/Cas system in bacteria
Doi:10.1038/nature11723
In a new study, a team of University of Toronto's Alan Davidson found that a single prophage in the Pseudomonas aeruginosa genome is resistant to a range of other types of phage. Therefore, they adjusted the P. aeruginosa CRISPR system in an attempt to understand how this prophage might affect the susceptibility of this bacterium to phage infection. They found that the IF-type CRISPR system of P. aeruginosa only blocked infection by certain types of phage. By searching for phage genomes, they found five unique genes, each with anti-CRISPR activity.
9. Harbin Dahuang Zhiwei Group's latest Nature! Reveal the molecular mechanism of the magic scissors "inhibition switch"
Doi:10.1038/nature22377
On April 28th, Prof. Huang Zhiwei from the School of Life Science, Harbin Institute of Technology published the topic "Anti-CRISPR Protein Inhibition of CRISPR-SpyCas9 Activity" on "Nature" (Structural basis of CRISPR-SpyCas9 inhibition). Research paper by an anti-CRISPR protein).
Earlier studies found that a class of anti-CRISPR genes, AcrIIA4, derived from Listeria monocytogenes proppage inhibited the gene-editing activity of SpyCas9 in cells. However, the molecular mechanism by which these Anti-CRISPR genes inhibit SpyCas9 activity is unclear.
In order to study whether AcrIIA2 or AcrIIA4 can directly bind to SpyCas9, the research team first established an in vitro biochemical research system. The study found that the Anti-CRISPR protein AcrIIA2 or AcrIIA4 directly binds to the SpyCas9-sgRNA complex. Interestingly, AcrIIA2 or AcrIIA4 only binds to sgRNA. SpyCas9 interacts and does not interact with SpyCas9 alone; further experiments have shown that AcrIIA2 or AcrIIA4 can directly inhibit SpyCas9-mediated DNA cleavage of the target DNA.
To investigate the molecular mechanism by which AcrIIA4 directly inhibits SpyCas9 activity, the group purified the SpyCas9-sgRNA-AcrIIA4 complex and resolved the crystal structure of the SpyCas9-sgRNA-AcrIIA4 complex by structural biology studies. The complex revealed AcrIIA4 protein. The conformation is a new fold that combines the grooved regions formed by the three domains of CTD, TOPO and RuvC of SpyCas9. This groove region is the binding site for the substrate PAM DNA on SpyCas9; in addition, the Loop in front of the β1 folded sheet from AcrIIA4 and the RuvC active site also enhance the inhibitory effect of AcrIIA4 on SpyCas9. The above structural observations showed that AcrIIA4 and substrate PAM DNA competitively bind to SpyCas9, and AcrIIA4 has a stronger affinity than the substrate PAM DNA. The results of structural observation further support the results of structural observation. Subsequent biochemical experiments further confirmed that AcrIIA4 binds to SpyCas9 and inhibits substrate PAM DNA binding to SpyCas9, thereby antagonizing the activity of SpyCas9.
10.Cell: The first time the "close switch" of the CRISPR/Cas9 gene editing was discovered.
Doi:10.1016/j.cell.2016.11.017
The CRISPR/Cas9 genome editor is rapidly eliciting changes in biomedical research, but this new technology has not been very accurate to date. This technique can inadvertently produce excessive or unwanted changes in the genome, resulting in off-target mutations, thereby limiting its safety and efficacy in therapeutic applications.
Now, in a new study, researchers from the University of Massachusetts Medical School and the University of Toronto in Canada found the first known CRISPR/Cas9 activity "off switch" to provide better control for the CRISPR/Cas9 editor.
Dr. Erik J. Sontheimer, Professor of the Institute of RNA Therapy at the University of Massachusetts Medical School, Dr. Alan Davidson, Professor of Molecular Genetics at the University of Toronto, and Dr. Karen Maxwell, Assistant Professor of Biochemistry at the University of Toronto, identified three naturally occurring proteins that inhibit Cas9 nuclease. These proteins, called CRISPR-resistant proteins, block the ability of Cas9 to cleave DNA.
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