C2c1 is a type V-B CRISPR-Cas system dual-RNA-guided DNA endonuclease. Here, we report the crystal structure of Alicyclobacillus acidoterrestris C2c1 in complex with a chimeric single-molecule guide ...RNA (sgRNA). AacC2c1 exhibits a bi-lobed architecture consisting of a REC and NUC lobe. The sgRNA scaffold forms a tetra-helical structure, distinct from previous predictions. The crRNA is located in the central channel of C2c1, and the tracrRNA resides in an external surface groove. Although AacC2c1 lacks a PAM-interacting domain, our analysis revealed that the PAM duplex has a similar binding position found in Cpf1. Importantly, C2c1-sgRNA system is highly sensitive to single-nucleotide mismatches between guide RNA and target DNA. The resulting reduction in off-target cleavage renders C2c1 a valuable addition to the current arsenal of genome-editing tools. Together, our findings indicate that sgRNA assembly is achieved through a mechanism distinct from that reported previously for Cas9 or Cpf1 endonucleases.
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•Crystal structure of Alicyclobacillus acidoterrestris C2c1-sgRNA complex•Mechanistic insights into dual-RNA-guided, C2c1-catalyzed, staggered dsDNA breaks•High mismatch sensitivity of C2c1 reduces off-target cleavage•Join 2/4-truncated sgRNA-guided DNA cleavage similar to that of wild-type sgRNA
Liu et al. report the structure of C2c1-a type V-B CRISPR-Cas endonuclease, in complex with a chimeric single guide RNA, providing insights into the high specificity of DNA cleavage by C2c1. C2c1’s low off-target rate makes it a valuable addition to the current arsenal of gene-editing tools.
Clustered regularly interspaced short palindromic repeats (CRISPRs)‐CRISPR‐associated protein systems are bacterial and archaeal defense mechanisms against invading elements such as phages and ...viruses. To overcome these defense systems, phages and viruses have developed inhibitors called anti‐CRISPRs (Acrs) that are capable of inhibiting the host CRISPR‐Cas system via different mechanisms. Although the inhibitory mechanisms of AcrIIC1, AcrIIC2, and AcrIIC3 have been revealed, the inhibitory mechanisms of AcrIIC4 and AcrIIC5 have not been fully understood and structural data are unavailable. In this study, we elucidated the crystal structure of Type IIC anti‐CRISPR protein, AcrIIC4. Our structural analysis revealed that AcrIIC4 exhibited a helical bundle fold comprising four helixes. Further biochemical and biophysical analyses showed that AcrIIC4 formed a monomer in solution, and monomeric AcrIIC4 directly interacted with Cas9 and Cas9/sgRNA complex. Discovery of the structure of AcrIIC4 and their interaction mode on Cas9 will help us elucidate the diversity in the inhibitory mechanisms of the Acr protein family.
PDB Code(s): 7F7P;
The RNA-guided endonuclease Cas9 generates a double-strand break at DNA target sites complementary to the guide RNA and has been harnessed for the development of a variety of new technologies, such ...as genome editing. Here, we report the crystal structures of Campylobacter jejuni Cas9 (CjCas9), one of the smallest Cas9 orthologs, in complex with an sgRNA and its target DNA. The structures provided insights into a minimal Cas9 scaffold and revealed the remarkable mechanistic diversity of the CRISPR-Cas9 systems. The CjCas9 guide RNA contains a triple-helix structure, which is distinct from known RNA triple helices, thereby expanding the natural repertoire of RNA triple helices. Furthermore, unlike the other Cas9 orthologs, CjCas9 contacts the nucleotide sequences in both the target and non-target DNA strands and recognizes the 5′-NNNVRYM-3′ as the protospacer-adjacent motif. Collectively, these findings improve our mechanistic understanding of the CRISPR-Cas9 systems and may facilitate Cas9 engineering.
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•Crystal structure of C. jejuni Cas9 bound to guide RNA and target DNA•The C. jejuni guide RNA contains a triple-helix architecture•C. jejuni Cas9 recognizes both strands in the 5′-NNNVRYM-3′ PAM duplex•Structural and mechanistic diversity among the orthologous CRISPR-Cas9 systems
Yamada et al. report the crystal structures of the minimal Cas9 from Campylobacter jejuni in complex with crRNA and its target DNA. The structures reveal the remarkable diversity in the guide RNA architecture and the PAM recognition among the CRISPR-Cas9 systems.
Class 2 CRISPR-Cas nucleases are programmable genome editing tools with promising applications in human health and disease. However, DNA cleavage at off-target sites that resemble the target sequence ...is a pervasive problem that remains poorly understood mechanistically. Here, we use quantitative kinetics to dissect the reaction steps of DNA targeting by Acidaminococcus sp Cas12a (also known as Cpf1). We show that Cas12a binds DNA tightly in two kinetically separable steps. Protospacer-adjacent motif (PAM) recognition is followed by rate-limiting R-loop propagation, leading to inevitable DNA cleavage of both strands. Despite functionally irreversible binding, Cas12a discriminates strongly against mismatches along most of the DNA target sequence. This result implies substantial reversibility during R-loop formation—a late transition state—and defies common descriptions of a “seed” region. Our results provide a quantitative basis for the DNA cleavage patterns measured in vivo and observations of greater reported target specificity for Cas12a than for the Cas9 nuclease.
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•DNA target binding by CRISPR-Cas12a is rate limiting for DNA cleavage•Multiple cleavage sites are present on each DNA strand•Specificity against mismatches suggests a late transition state for R-loop formation•Data explain in vivo cleavage patterns by Cas12a and differences from Cas9
Strohkendl et al. dissect DNA binding and cleavage by CRISPR-Cas12a. They show that binding is functionally irreversible, yet Cas12a discriminates against mismatches with the target DNA extending beyond a seed region. These results suggest that R-loop propagation is readily reversible, enabling Cas12a to select DNA sequences more precisely than Cas9.
Protein-based Cas9 in vivo gene editing therapeutics have practical limitations owing to their instability and low efficacy. To overcome these obstacles and improve stability, we designed a ...nanocarrier primarily consisting of lecithin that can efficiently target liver disease and encapsulate complexes of Cas9 with a single-stranded guide RNA (sgRNA) ribonucleoprotein (Cas9-RNP) through polymer fusion self-assembly.
In this study, we optimized an sgRNA sequence specifically for dipeptidyl peptidase-4 gene (DPP-4) to modulate the function of glucagon-like peptide 1. We then injected our nanocarrier Cas9-RNP complexes directly into type 2 diabetes mellitus (T2DM) db/db mice, which disrupted the expression of DPP-4 gene in T2DM mice with remarkable efficacy. The decline in DPP-4 enzyme activity was also accompanied by normalized blood glucose levels, insulin response, and reduced liver and kidney damage. These outcomes were found to be similar to those of sitagliptin, the current chemical DPP-4 inhibition therapy drug which requires recurrent doses.
Our results demonstrate that a nano-liposomal carrier system with therapeutic Cas9-RNP has great potential as a platform to improve genomic editing therapies for human liver diseases.
Summary
Over the last three decades, the development of new genome editing techniques, such as ODM, TALENs, ZFNs and the CRISPR‐Cas system, has led to significant progress in the field of plant and ...animal breeding. The CRISPR‐Cas system is the most versatile genome editing tool discovered in the history of molecular biology because it can be used to alter diverse genomes (e.g. genomes from both plants and animals) including human genomes with unprecedented ease, accuracy and high efficiency. The recent development and scope of CRISPR‐Cas system have raised new regulatory challenges around the world due to moral, ethical, safety and technical concerns associated with its applications in pre‐clinical and clinical research, biomedicine and agriculture. Here, we review the art, applications and potential risks of CRISPR‐Cas system in genome editing. We also highlight the patent and ethical issues of this technology along with regulatory frameworks established by various nations to regulate CRISPR‐Cas‐modified organisms/products.
•SARS-CoV-2 has infected millions people worldwide with numerous deaths since December 2019.•Type V and VI CRISPR enzymes are efficient agents for diagnosis and combating single-stranded RNA viruses ...such as SARS-CoV-2.•DETECTR and SHERLOCK represent rapid, specific and efficient diagnostic platforms for point of care detection of SARS-CoV-2.•PAC-MAN is a SARS-CoV-2 plausible therapeutic approach which pave the way to use CRISPR systems as robust antivirals.
Type V and VI CRISPR enzymes are RNA-guided, DNA and RNA-targeting effectors that allow specific gene knockdown. Cas12 and Cas13 are CRISPR proteins that are efficient agents for diagnosis and combating single-stranded RNA (ssRNA) viruses. The programmability of these proteins paves the way for the detection and degradation of RNA viruses by targeting RNAs complementary to its CRISPR RNA (crRNA). Approximately two-thirds of viruses causing diseases contain ssRNA genomes. The Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) has caused the outbreak of the coronavirus disease 2019 (COVID-19), which has infected more than 88 million people worldwide with near 2 million deaths since December 2019. Thus, accurate and rapid diagnostic and therapeutic tools are essential for early detection and treatment of this widespread infectious disease. For us, the CRISPR based platforms seem to be a plausible new approach for an accurate detection and treatment of SARS-CoV-2.
In this review, we talk about Cas12 and Cas13 CRISPR systems and their applications in diagnosis and treatment of RNA virus mediated diseases. In continue, the SARS-CoV-2 pathogenicity, and its conventional diagnostics and antivirals will be discussed. Moreover, we highlight novel CRISPR based diagnostic platforms and therapies for COVID-19. We also discuss the challenges of diagnostic CRISPR based platforms as well as clarifying the proposed solution for high efficient selective in vivo delivery of CRISPR components into SARS-CoV-2-infected cells.
Clostridium butyricum has been widely used as a probiotic for humans and food animals. However, the mechanisms of beneficial effects of C. butyricum on the host remain poorly understood, largely due ...to the lack of high‐throughput genome engineering tools. Here, we report the exploitation of heterologous Type II CRISPR‐Cas9 system and endogenous Type I‐B CRISPR‐Cas system in probiotic C. butyricum for seamless genome engineering. Although successful genome editing was achieved in C. butyricum when CRISPR‐Cas9 system was employed, the expression of toxic cas9 gene result in really poor transformation, spurring us to develop an easy‐applicable and high‐efficient genome editing tool. Therefore, the endogenous Type I‐B CRISPR‐Cas machinery located on the megaplasmid of C. butyricum was co‐opted for genome editing. In vivo plasmid interference assays identified that ACA and TAA were functional protospacer adjacent motif sequences needed for site‐specific CRISPR attacking. Using the customized endogenous CRISPR‐Cas system, we successfully deleted spo0A and aldh genes in C. butyricum, yielding an efficiency of up to 100%. Moreover, the conjugation efficiency of endogenous CRISPR‐Cas system was dramatically enhanced due to the precluding expression of cas9. Altogether, the two approaches developed herein remarkably expand the existing genetic toolbox available for investigation of C. butyricum.
The year 2020 witnessed an unpredictable pandemic situation due to novel coronavirus (COVID-19) outbreaks. This condition can be more severe if the patient has comorbidities. Failure of viable ...treatment for such viral infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is due to lack of identification. Thus, modern and productive biotechnology-based tools are being used to manipulate target genes by introducing the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas (CRISPR-associated) system. Moreover, it has now been used as a tool to inhibit viral replication. Hence, it can be hypothesized that the CRISPR/Cas system can be a viable tool to target both the SARS-CoV-2 genome with specific target RNA sequence and host factors to destroy the SARS-CoV-2 community via inhibition of viral replication and infection. Moreover, comorbidities and COVID-19 escalate the rate of mortality globally, and as a result, we have faced this pandemic. CRISPR/Cas-mediated genetic manipulation to knockdown viral sequences may be a preventive strategy against such pandemic caused by SARS-CoV-2. Furthermore, prophylactic antiviral CRISPR in human cells (PAC-MAN) along with CRISPR/Cas13d efficiently degrades the specific RNA sequence to inhibit viral replication. Therefore, we suggest that CRISPR/Cas system with PAC-MAN could be a useful tool to fight against such a global pandemic caused by SARS-CoV-2. This is an alternative preventive approach of management against the pandemic to destroy the target sequence of RNA in SARS-CoV-2 by viral inhibition.
CRISPR-Cas systems are bacterial anti-viral systems, and phages use anti-CRISPR proteins (Acrs) to inactivate these systems. Here, we report a novel mechanism by which AcrIF11 inhibits the type I-F ...CRISPR system. Our structural and biochemical studies demonstrate that AcrIF11 functions as a novel mono-ADP-ribosyltransferase (mART) to modify N250 of the Cas8f subunit, a residue required for recognition of the protospacer-adjacent motif, within the crRNA-guided surveillance (Csy) complex from Pseudomonas aeruginosa. The AcrIF11-mediated ADP-ribosylation of the Csy complex results in complete loss of its double-stranded DNA (dsDNA) binding activity. Biochemical studies show that AcrIF11 requires, besides Cas8f, the Cas7.6f subunit for binding to and modifying the Csy complex. Our study not only reveals an unprecedented mechanism of type I CRISPR-Cas inhibition and the evolutionary arms race between phages and bacteria but also suggests an approach for designing highly potent regulatory tools in the future applications of type I CRISPR-Cas systems.
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•Crystal structure of AcrIF11 suggests that it resembles an ADP-ribosyltransferase•AcrIF11 specifically ADP-ribosylates a key residue in the PAM-recognition loop•AcrIF11-mediated ADP-ribosylation of the Csy complex prevents dsDNA binding•AcrIF11 requires the Cas7.6f subunit for binding to and modifying the Csy complex
Niu et al. report structural and biochemical data that reveal the molecular mechanism of AcrIF11-mediated inhibition of the type I-F CRISPR system. AcrIF11 specifically ADP-ribosylates a key residue in the PAM-recognition loop of the type I-F Cascade complex, thereby inhibiting its DNA binding activity and inactivating the CRISPR system.
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