Anti-CRISPR (Acr) proteins are encoded by phages and other mobile genetic elements and inhibit host CRISPR-Cas immunity using versatile strategies. AcrIIC4 is a broad-spectrum Acr that inhibits the ...type II-C CRISPR-Cas9 system in several species by an unknown mechanism. Here, we determined a series of structures of
Cas9 (HpaCas9)-sgRNA in complex with AcrIIC4 and/or target DNA, as well as the crystal structure of AcrIIC4 alone. We found that AcrIIC4 resides in the crevice between the REC1 and REC2 domains of HpaCas9, where its extensive interactions restrict the mobility of the REC2 domain and prevent the unwinding of target double-stranded (ds) DNA at the PAM-distal end. Therefore, the full-length guide RNA:target DNA heteroduplex fails to form in the presence of AcrIIC4, preventing Cas9 nuclease activation. Altogether, our structural and biochemical studies illuminate a unique Acr mechanism that allows DNA binding to the Cas9 effector complex but blocks its cleavage by preventing R-loop formation, a key step supporting DNA cleavage by Cas9.
Although CRISPR-Cas9/Cpf1 have been employed as powerful genome engineering tools, heterologous CRISPR-Cas9/Cpf1 are often difficult to introduce into bacteria and archaea due to their severe ...toxicity. Since most prokaryotes harbor native CRISPR-Cas systems, genome engineering can be achieved by harnessing these endogenous immune systems. Here, we report the exploitation of Type I-B CRISPR-Cas of Clostridium tyrobutyricum for genome engineering. In silico CRISPR array analysis and plasmid interference assay revealed that TCA or TCG at the 5′-end of the protospacer was the functional protospacer adjacent motif (PAM) for CRISPR targeting. With a lactose inducible promoter for CRISPR array expression, we significantly decreased the toxicity of CRISPR-Cas and enhanced the transformation efficiency, and successfully deleted spo0A with an editing efficiency of 100%. We further evaluated effects of the spacer length on genome editing efficiency. Interestingly, spacers ≤ 20 nt led to unsuccessful transformation consistently, likely due to severe off-target effects; while a spacer of 30–38 nt is most appropriate to ensure successful transformation and high genome editing efficiency. Moreover, multiplex genome editing for the deletion of spo0A and pyrF was achieved in a single transformation, with an editing efficiency of up to 100%. Finally, with the integration of the alcohol dehydrogenase gene (adhE1 or adhE2) to replace cat1 (the key gene responsible for butyrate production and previously could not be deleted), two mutants were created for n-butanol production, with the butanol titer reached historically record high of 26.2 g/L in a batch fermentation. Altogether, our results demonstrated the easy programmability and high efficiency of endogenous CRISPR-Cas. The developed protocol herein has a broader applicability to other prokaryotes containing endogenous CRISPR-Cas systems. C. tyrobutyricum could be employed as an excellent platform to be engineered for biofuel and biochemical production using the CRISPR-Cas based genome engineering toolkit.
•The native Type I-B CRISPR-Cas of C. tyrobutyricum was harnessed for genome editing.•Off-target effects were revealed, and appropriate spacer length was suggested.•Single and multiplex genome editing were achieved with efficiencies of up to 100%.•Carbon flow was redirected to butanol by replacement of cat1 gene with adhE1/adhE2.•High level butanol (26.2 g/L) was obtained in batch fermentation at low temperature.
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) system represents, in prokaryotes, an adaptive and inheritable immune response against invading ...DNA. The discovery of anti-CRISPR proteins (Acrs), which are inhibitors of CRISPR-Cas, mainly encoded by phages and prophages, showed a co-evolution history between prokaryotes and phages. In the past decade, the CRISPR-Cas systems together with the corresponding Acrs have been turned into a genetic-engineering tool. Among the six types of CRISPR-Cas characterized so far, type II CRISPR-Cas system is the most popular in biotechnology. Here, we discuss about the discovery, the reported inhibitory mechanisms, and the applications in both gene editing and gene transcriptional regulation of type II Acrs. Moreover, we provide insights into future potential research and feasible applications.
Tuberculosis (TB), a leading cause of mortality globally, is a chronic infectious disease caused by Mycobacterium tuberculosis that primarily infiltrates the lung. The mature crRNAs in M. ...tuberculosis transcribed from the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) locus exhibit an atypical structure featured with 5′ and 3′ repeat tags at both ends of the intact crRNA, in contrast to typical Type-III-A crRNAs that possess 5′ repeat tags and partial crRNA sequences. However, this structural peculiarity particularly concerning the specific binding characteristics of the 3′ repeat end within the mature crRNA within the Csm complex, has not been comprehensively elucidated. Here, our Mycobacteria CRISPR-Csm complexes structure represents the largest Csm complex reported to date. It incorporates an atypical Type-III-A CRISPR RNA (crRNA) (46 nt) with 5′ 8-nt and 3′ 4-nt repeat sequences in the stoichiometry of Mycobacteria Csm1125364151. The PAM-independent single-stranded RNAs (ssRNAs) are the most suitable substrate for the Csm complex. The 3′-repeat end trimming of mature crRNA was not necessary for its cleavage activity in Type-III-A Csm complex. Our work broadens our understanding of the Type-III-A Csm complex and identifies another mature crRNA processing mechanism in the Type-III-A CRISPR-Cas system based on structural biology.
New tools for genetic manipulation of
are needed for the development of new drug regimens and vaccines aimed at curing tuberculosis infections. Clustered regularly interspaced short palindromic ...repeat (CRISPR)-CRISPR-associated protein (Cas) systems generate a highly specific double-strand break at the target site that can be repaired via nonhomologous end joining (NHEJ), resulting in the desired genome alteration. In this study, we first improved the NHEJ repair pathway and developed a CRISPR-Cas-mediated genome-editing method that allowed us to generate markerless deletion in
,
, and
Then, we demonstrated that this system could efficiently achieve simultaneous generation of double mutations and large-scale genetic mutations in
Finally, we showed that the strategy we developed can also be used to facilitate genome editing in
The global health impact of
necessitates the development of new genetic tools for its manipulation, to facilitate the identification and characterization of novel drug targets and vaccine candidates. Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) genome editing has proven to be a powerful genetic tool in various organisms; to date, however, attempts to use this approach in
have failed. Here, we describe a genome-editing tool based on CRISPR cleavage and the nonhomologous end-joining (NHEJ) repair pathway that can efficiently generate deletion mutants in
More importantly, this system can generate simultaneous double mutations and large-scale genetic mutations in this species. We anticipate that this CRISPR-NHEJ-assisted genome-editing system will be broadly useful for research on mycobacteria, vaccine development, and drug target profiling.
CRISPR-Cas systems adapt their immunological memory against their invaders by integrating short DNA fragments into clustered regularly interspaced short palindromic repeat (CRISPR) loci. While Cas1 ...and Cas2 make up the core machinery of the CRISPR integration process, various class I and II CRISPR-Cas systems encode Cas4 proteins for which the role is unknown. Here, we introduced the CRISPR adaptation genes cas1, cas2, and cas4 from the type I-D CRISPR-Cas system of Synechocystis sp. 6803 into Escherichia coli and observed that cas4 is strictly required for the selection of targets with protospacer adjacent motifs (PAMs) conferring I-D CRISPR interference in the native host Synechocystis. We propose a model in which Cas4 assists the CRISPR adaptation complex Cas1-2 by providing DNA substrates tailored for the correct PAM. Introducing functional spacers that target DNA sequences with the correct PAM is key to successful CRISPR interference, providing a better chance of surviving infection by mobile genetic elements.
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•Cas4 facilitates the integration of PAM-compatible spacers•Spacer length variation is dictated by Cas1-2•Cas4 shortens spacer length•Cas4-selected PAMs license type I-D CRISPR interference
Kieper et al. demonstrate that the ubiquitous protein Cas4 assists Cas1 and Cas2 in the selection of new CRISPR spacers with a PAM licensing efficient CRISPR interference.
CRISPR-Cas systems have been co-opted by Tn7-like transposable elements to direct RNA-guided transposition. Type V-K CRISPR-associated transposons rely on the concerted activities of the ...pseudonuclease Cas12k, the AAA+ ATPase TnsC, the Zn-finger protein TniQ, and the transposase TnsB. Here we present a cryo-electron microscopic structure of a target DNA-bound Cas12k-transposon recruitment complex comprised of RNA-guided Cas12k, TniQ, a polymeric TnsC filament and, unexpectedly, the ribosomal protein S15. Complex assembly, mediated by a network of interactions involving the guide RNA, TniQ, and S15, results in R-loop completion. TniQ contacts two TnsC protomers at the Cas12k-proximal filament end, likely nucleating its polymerization. Transposition activity assays corroborate our structural findings, implying that S15 is a bona fide component of the type V crRNA-guided transposon machinery. Altogether, our work uncovers key mechanistic aspects underpinning RNA-mediated assembly of CRISPR-associated transposons to guide their development as programmable tools for site-specific insertion of large DNA payloads.
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•Cryo-EM structure of CRISPR RNA-guided Cas12k-transposon recruitment complex•TniQ interacts with tracrRNA and facilitates R-loop completion•TniQ connects Cas12k with TnsC ATPase filament end on target DNA•Ribosomal protein S15 is a bona fide component of type V CRISPR-associated transposons
Cryo-EM structure of a type V CRISPR-associated transposon recruitment complex reveals the mechanism linking the RNA-guided pseudonuclease Cas12k and the transposition machinery, and the involvement of a ribosomal protein in the assembly, providing a framework for the development of novel programmable gene-insertion technologies.
The CRISPR-Cas systems have shown tremendous promise as heterologous tools for genome editing in various prokaryotes. However, the perturbation of DNA homeostasis and the inherent toxicity of ...Cas9/12a proteins could easily lead to cell death, which led to the development of endogenous CRISPR-Cas systems. Programming the widespread endogenous CRISPR-Cas systems for in situ genome editing represents a promising tool in prokaryotes, especially in genetically intractable species. Here, this review briefly summarizes the advances of endogenous CRISPR-Cas-mediated genome editing, covering aspects of establishing and optimizing the genetic tools. In particular, this review presents the application of different types of endogenous CRISPR-Cas tools for strain engineering, including genome editing and genetic regulation. Notably, this review also provides a detailed discussion of the transposon-associated CRISPR-Cas systems, and the programmable RNA-guided transposition using endogenous CRISPR-Cas systems to enable editing of microbial communities for understanding and control. Therefore, they will be a powerful tool for targeted genetic manipulation. Overall, this review will not only facilitate the development of standard genetic manipulation tools for non-model prokaryotes but will also enable more non-model prokaryotes to be genetically tractable.
CRISPR-Cas-mediated diagnostics Li, Luoxi; Shen, Guanwang; Wu, Min ...
Trends in biotechnology (Regular ed.),
November 2022, 2022-11-00, 20221101, Letnik:
40, Številka:
11
Journal Article
Recenzirano
An ideal molecular diagnostic method should be sensitive, specific, low cost, rapid, portable, and easy to operate. Traditional nucleic acid detection methods based mainly on PCR technology have not ...only high sensitivity and specificity, but also some limitations, such as the need for expensive equipment and skilled technicians, being both time and labor intensive, and difficult to implement in some regions. However, with the continuous development of CRISPR-Cas technology and its application in molecular diagnosis, new approaches have been used for the construction of molecular diagnostic systems. In this review, we discuss recent advances in CRISPR-based molecular diagnostic technologies and highlight the revolution they bring to the field of molecular diagnostics.
CRISPR-Cas-mediated diagnostics can detect various pathogens, cancer biomarkers, and SNPs with high specificity, speed, low cost, and portability.The combination of Cas proteins with biosensors, biochips, biomagnetic beads, isothermal amplification, lateral flow, protein aptamers, and other technologies has resulted in the development of new diagnostic methods.Multiplexed CRISPR nucleic acid detection systems complete the detection of 169 viruses for diagnosing different diseases in a single sample, with the help of microfluidic technology.CRISPR-mediated antibody diagnosis technology can recognize thousands of antibodies in a sample simultaneously.The potential of CRISPR-Cas biosensing technologies is inspiring researchers to develop next-generation diagnostics platforms.
The CRISPR-Cas systems in prokaryotes are RNA-guided immune systems that target and deactivate foreign nucleic acids. A typical CRISPR-Cas system consists of a CRISPR array of repeat and spacer ...units, and a locus of cas genes. The CRISPR and the cas locus are often located next to each other in the genomes. However, there is no quantitative estimate of the co-location. In addition, ad-hoc studies have shown that some non-CRISPR genomic elements contain repeat-spacer-like structures and are mistaken as CRISPRs.
Using available genome sequences, we observed that a significant number of genomes have isolated cas loci and/or CRISPRs. We found that 11%, 22% and 28% of the type I, II and III cas loci are isolated (without CRISPRs in the same genomes at all or with CRISPRs distant in the genomes), respectively. We identified a large number of genomic elements that superficially reassemble CRISPRs but don't contain diverse spacers and have no companion cas genes. We called these elements false-CRISPRs and further classified them into groups, including tandem repeats and Staphylococcus aureus repeat (STAR)-like elements.
This is the first systematic study to collect and characterize false-CRISPR elements. We demonstrated that false-CRISPRs could be used to reduce the false annotation of CRISPRs, therefore showing them to be useful for improving the annotation of CRISPR-Cas systems.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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