Search

The discovery1,2and the pioneering applications3of CRISPR-Cas effector complexes have provided powerful gene-editing tools. The effector complexes are guided to the targeted genomic locus by the complementarity of their CRISPR RNA (crRNA)4,5. Recognition of double-stranded DNA targets proceeds via DNA unwinding and base-pairing between crRNA and the DNA target strand resulting in the formation of an R-loop structure5,6. Full R-loop formation is the prerequisite for the subsequent DNA cleavage. While the CRISPR-Cas technology is easy to use, efficient and highly versatile, therapeutic applications are hampered by the off-target effects due to the recognition of unintended sequences with multiple mismatches7. This process is still poorly understood on a mechanistic level8,9. Particularly, the lack of insight into the energetics and dynamics of the R-loop formation hinders a direct modelling of the R-loop formation for off-target prediction.Here we set up ultrafast DNA unwinding experiments based on plasmonic DNA nanorotors to follow the R-loop formation by the Cascade effector complex in real time, close to base pair resolution. We directly resolve a weak global downhill bias of the energy landscape of the forming R-loop followed by a steep uphill bias for the final base pairs. We furthermore show a modulation of the landscape by base flips and mismatches. These data provide that Cascade-mediated R-loop formation occurs on short time scales in single base pair steps of sub-millisecond duration, but on longer time scales in six–base pair intermediate steps in agreement with the structural periodicity of the crRNA-DNA hybrid. We expect that the knowledge about the energy landscapes of R-loop formation of CRISPR-Cas effector complexes will pave the way for a detailed understanding and prediction of off-target recognition10.

CRISPR-Cas effector complexes enable the defense against foreign nucleic acids and have recently been exploited as molecular tools for precise genome editing at a target locus. To bind and cleave their target, the CRISPR-Cas effectors first have to interrogate the entire genome for the presence of a matching sequence. Matching is achieved by base-pairing between the crRNA of the complexes and the DNA target strand such that an R-loop is formed. R-loop formation starts at a specific PAM motif and progresses reversibly in single base-pair steps until mismatches stop further progression or until the full target is recognized and destroyed. The reversible nature of this process entails that even a fully matching target should only become recognized with a low probability per target encounter. The details of this process, which directly affect the effectiveness of the target search, remain unresolved.Here we dissect the target search process of the Type I CRISPR-Cas complex Cascade by simultaneously monitoring DNA binding and R-loop formation by the complex. We directly quantify the low target recognition probabilities and show that they increase with increasing negative supercoiling. Furthermore, we demonstrate that Cascade uses a combination of three-dimensional and limited one-dimensional diffusion along the DNA contour for its target search. The latter allows for rapidly scanning the PAM sequences in a given region and, importantly, significantly increasing the overall efficiency of the target search by repeatedly revisiting the sites. Overall we show that target search and target recognition are tightly linked and that DNA supercoiling and limited 1D diffusion need to be considered when understanding target recognition and target search by CRISPR-Cas enzymes and engineering more efficient and precise variants.

BackgroundCRISPR-Cas systems, which provide adaptive immunity against foreign nucleic acids in prokaryotes, can serve as useful molecular tools for multiple applications in genome engineering. Diverse CRISPR-Cas systems originating from distinct prokaryotes function through a common mechanism involving the assembly of small crRNA molecules and Cas proteins into a ribonucleoprotein (RNP) effector complex, and formation of an R-loop structure upon binding to the target DNA. Extensive research on the I-E subtype established the prototypical mechanism of DNA interference in type I systems, where the coordinated action of a ribonucleoprotein Cascade complex and Cas3 protein destroys foreign DNA. However, diverse protein composition between type I subtypes suggests differences in the mechanism of DNA interference that could be exploited for novel practical applications that call for further exploration of these systems.ResultsHere we examined the mechanism of DNA interference provided by the type I-F1 system fromAggregatibacter actinomycetemcomitansD7S-1 (Aa). We show that functional Aa-Cascade complexes can be assembled not only with WT spacer of 32 nt but also with shorter or longer (14–176 nt) spacers. All complexes guided by the spacer bind to the target DNA sequence (protospacer) forming an R-loop when a C or CT protospacer adjacent motif (PAM) is present immediately upstream the protospacer (at −1 or −2,−1 position, respectively). The range of spacer and protospacer complementarity predetermine the length of the R-loop; however, only R-loops of WT length or longer trigger the nuclease/helicase Cas2/3, which initiates ATP-dependent unidirectional degradation at the PAM-distal end of the WT R-loop. Meanwhile, truncation of the WT R-loop at the PAM-distal end abolishes Cas2/3 cleavage.ConclusionsWe provide a comprehensive characterisation of the DNA interference mechanism in the type I-F1 CRISPR-Cas system, which is different from the type I-E in a few aspects. First, DNA cleavage initiation, which usually happens at the PAM-proximal end in type I-E, is shifted to the PAM-distal end of WT R-loop in the type I-F1. Second, the R-loop length controls on/off switch of DNA interference in the type I-F1, while cleavage initiation is less restricted in the type I-E. These results indicate that DNA interference in type I-F1 systems is governed through a checkpoint provided by the Cascade complex, which verifies the appropriate length for the R-loop.

Summary The multi-subunit type I CRISPR-Cas surveillance complex Cascade uses its crRNA to recognize dsDNA targets. Recognition involves DNA unwinding and base-pairing between the crRNA spacer region and a complementary DNA strand, resulting in formation of an R-loop structure. The modular Cascade architecture allows assembly of complexes containing crRNAs with altered spacer lengths that promise increased target specificity in emerging biotechnological applications. Here we produce type I-E Cascade complexes containing crRNAs with up to 57-nt-long spacers. We show that these complexes form R-loops corresponding to the designed target length, even for the longest spacers tested. Furthermore, the complexes can bind their targets with much higher affinity compared with the wild-type form. However, target recognition and the subsequent Cas3-mediated DNA cleavage do not require extended R-loops but already occur for wild-type-sized R-loops. These findings set important limits for specificity improvements of type I CRISPR-Cas systems., Graphical Abstract, Highlights • Cascades with elongated crRNA spacers support the formation of up to 57-bp-long R-loops • Target recognition and DNA degradation require only wild-type-sized, ∼30-bp-long R-loops • Complexes with elongated crRNAs bind targets with significantly increased affinity, Songailiene et al. show that engineered versions of Cascade with elongated crRNAs can base-pair with target DNA over 57 bp. However, target recognition only requires wild-type-sized base-pairing of ∼30-bp length. These findings define constraints for improving the specificity of these complexes in biotechnological applications.

Prokaryotes evolved numerous systems that defend against predation by bacteriophages. In addition to well-known restriction-modification and CRISPR-Cas immunity systems, many poorly characterized systems exist. One class of such systems, named BREX, consists of a putative phosphatase, a methyltransferase and four other proteins. A Bacillus cereus BREX system provides resistance to several unrelated phages and leads to modification of specific motif in host DNA. Here, we study the action of BREX system from a natural Escherichia coli isolate. We show that while it makes cells resistant to phage λ infection, induction of λ prophage from cells carrying BREX leads to production of viruses that overcome the defense. The induced phage DNA contains a methylated adenine residue in a specific motif. The same modification is found in the genome of BREX-carrying cells. The results establish, for the first time, that immunity to BREX system defense is provided by an epigenetic modification.

CRISPR‐Cas systems constitute an adaptive immune system that provides acquired resistance against phages and plasmids in prokaryotes. Upon invasion of foreign nucleic acids, some cells integrate short fragments of foreign DNA as spacers into the CRISPR locus to memorize the invaders and acquire resistance in the subsequent round of infection. This immunization step called adaptation is the least understood part of the CRISPR‐Cas immunity. We have focused here on the adaptation stage of Streptococcus thermophilus DGCC7710 type I‐E CRISPR4‐Cas (St4) system. Cas1 and Cas2 proteins conserved in nearly all CRISPR‐Cas systems are required for spacer acquisition. The St4 CRISPR‐Cas system is unique because the Cas2 protein is fused to an additional DnaQ exonuclease domain. Here, we demonstrate that St4 Cas1 and Cas2‐DnaQ form a multimeric complex, which is capable of integrating DNA duplexes with 3′‐overhangs (protospacers) in vitro. We further show that the DnaQ domain of Cas2 functions as a 3′–5′‐exonuclease that processes 3′‐overhangs of the protospacer to promote integration.

Bacteriophages (phages) infect bacteria in order to replicate and burst out of the host, killing the cell, when reproduction is completed. Thus, from a bacterial perspective, phages pose a persistent lethal threat to bacterial populations. Not surprisingly, bacteria evolved multiple defense barriers to interfere with nearly every step of phage life cycles. Phages respond to this selection pressure by counter-evolving their genomes to evade bacterial resistance. The antagonistic interaction between bacteria and rapidly diversifying viruses promotes the evolution and dissemination of bacteriophage-resistance mechanisms in bacteria. Recently, an adaptive microbial immune system, named clustered regularly interspaced short palindromic repeats (CRISPR) and which provides acquired immunity against viruses and plasmids, has been identified. Unlike the restriction–modification anti-phage barrier that subjects to cleavage any foreign DNA lacking a protective methyl-tag in the target site, the CRISPR–Cas systems are invader-specific, adaptive, and heritable. In this review, we focus on the molecular mechanisms of interference/immunity provided by different CRISPR–Cas systems. Electronic supplementary material The online version of this article (doi:10.1007/s00018-013-1438-6) contains supplementary material, which is available to authorized users.

Clustered regularly interspaced short palindromic repeat (CRISPR) is a recently discovered adaptive prokaryotic immune system that provides acquired immunity against foreign nucleic acids by utilizing small guide crRNAs (CRISPR RNAs) to interfere with invading viruses and plasmids. In Escherichia coli, Cas3 is essential for crRNA-guided interference with virus proliferation. Cas3 contains N-terminal HD phosphohydrolase and C-terminal Superfamily 2 (SF2) helicase domains. Here, we provide the first report of the cloning, expression, purification and in vitro functional analysis of the Cas3 protein of the Streptococcus thermophilus CRISPR4 (Ecoli subtype) system. Cas3 possesses a single-stranded DNA (ssDNA)-stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes. Cas3 also shows ATP-independent nuclease activity located in the HD domain with a preference for ssDNA substrates. To dissect the contribution of individual domains, Cas3 separation-of-function mutants (ATPase+/nuclease− and ATPase−/nuclease+) were obtained by site-directed mutagenesis. We propose that the Cas3 ATPase/helicase domain acts as a motor protein, which assists delivery of the nuclease activity to Cascade–crRNA complex targeting foreign DNA.

Cas3 is a signature protein of the type I CRISPR-Cas systems and typically contains HD phosphohydrolase and Superfamily 2 (SF2) helicase domains. In the type I CRISPR-Cas systems Cas3 functions as a slicer that provides foreign DNA degradation. Biochemical analysis indicate that Cas3 of the Streptococcus thermophilus DGCC7710 (St-Cas3) CRISPR4 system is a single-stranded DNA nuclease which also possesses a single-stranded DNA-stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes in 3' to 5' direction. The interplay between the nuclease and ATPase/helicase activities of St-Cas3 results in DNA degradation. Here, we describe assays for monitoring of St-Cas3 nuclease, ATPase and helicase activities in a stand-alone form and in the presence of the Cascade ribonucleoprotein complex. These assays can be easily adapted for biochemical analysis of Cas3 proteins from different microorganisms.

CRISPR-Cas systems provide adaptive immunity against invading genetic elements such as phages and plasmids by degrading the invader DNA. In type I CRISPR-Cas systems the intrinsic RNA component of the Cascade effector complex recognizes a complementary DNA target strand (protospacer) through base pairing while displacing the non-target strand of the duplex. The resulting RNA-DNA hybrid is called R-loop. R-loop establishment recruits the helicase-nuclease Cas3, which subsequently degrades the target DNA. Here we use single-molecule DNA supercoiling experiments to investigate the recognition and verification of the target sequence by Cascade. To this end we carefully explore the effect of mutations across the target. We observe the occurrence of intermediate R-loops which lengths correspond exactly to the distance of the mutation from the start of the target sequence. These intermediate R-loops are unstable and their stability directly correlates with their length. When point mutations are eventually overcome, a full R-loop forms and becomes “locked” through conformational changes of the Cascade complex. These observations provide direct evidence for a directional R-loop formation and suggest a dynamic target scanning by Cascade, mediated through R-loop intermediates. Early encountered mutations challenge R-loop propagation and cause its collapse while distal mutations enable more stable and longer lived R-loops to overcome them. The target validation takes place exclusively at the end of the sequence when full R-loop formation induces conformational changes that initiate DNA degradation. Such a dynamic and directional R-loop propagation scheme offers several advantages for suitable homologous target recognition: (i) the complex spends little time on wrong targets that from the beginning on carry mismatches; (ii) however it supports that point mutations can be tolerated thus precluding invading DNA to escape degradation through protospacer mutations.

SummaryCRISPR-Cas systems provide bacteria and archaea with adaptive immunity against foreign nucleic acids. In type I CRISPR-Cas systems, invading DNA is detected by a large ribonucleoprotein surveillance complex called Cascade. The crRNA component of Cascade is used to recognize target sites in foreign DNA (protospacers) by formation of an R-loop driven by base-pairing complementarity. Using single-molecule supercoiling experiments with near base-pair resolution, we probe here the mechanism of R-loop formation and detect short-lived R-loop intermediates on off-target sites bearing single mismatches. We show that R-loops propagate directionally starting from the protospacer-adjacent motif (PAM). Upon reaching a mismatch, R-loop propagation stalls and collapses in a length-dependent manner. This unambiguously demonstrates that directional zipping of the R-loop accomplishes efficient target recognition by rapidly rejecting binding to off-target sites with PAM-proximal mutations. R-loops that reach the protospacer end become locked to license DNA degradation by the auxiliary Cas3 nuclease/helicase without further target verification.

Cas3 is a signature protein of the type I CRISPR-Cas systems and typically contains HD phosphohydrolase and Superfamily 2 (SF2) helicase domains. In the type I CRISPR-Cas systems Cas3 functions as a slicer that provides foreign DNA degradation. Biochemical analysis indicate that Cas3 of the Streptococcus thermophilus DGCC7710 (St-Cas3) CRISPR4 system is a single-stranded DNA nuclease which also possesses a single-stranded DNA-stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes in 3' to 5' direction. The interplay between the nuclease and ATPase/helicase activities of St-Cas3 results in DNA degradation. Here, we describe assays for monitoring of St-Cas3 nuclease, ATPase and helicase activities in a stand-alone form and in the presence of the Cascade ribonucleoprotein complex. These assays can be easily adapted for biochemical analysis of Cas3 proteins from different microorganisms.

Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems protect bacteria and archaea from infection by viruses and plasmids. Central to this defense is a ribonucleoprotein complex that produces RNA-guided cleavage of foreign nucleic acids. In DNA-targeting CRISPR-Cas systems, the RNA component of the complex encodes target recognition by forming a site-specific hybrid (R-loop) with its complement (protospacer) on an invading DNA while displacing the noncomplementary strand. Subsequently, the R-loop structure triggers DNA degradation. Although these reactions have been reconstituted, the exact mechanism of R-loop formation has not been fully resolved. Here, we use single-molecule DNA supercoiling to directly observe and quantify the dynamics of torque-dependent R-loop formation and dissociation for both Cascade- and Cas9-based CRISPR-Cas systems. We find that the protospacer adjacent motif (PAM) affects primarily the R-loop association rates, whereas protospacer elements distal to the PAM affect primarily R-loop stability. Furthermore, Cascade has higher torque stability than Cas9 by using a conformational locking step. Our data provide direct evidence for directional R-loop formation, starting from PAM recognition and expanding toward the distal protospacer end. Moreover, we introduce DNA supercoiling as a quantitative tool to explore the sequence requirements and promiscuities of orthogonal CRISPR-Cas systems in rapidly emerging gene-targeting applications.

Clustered regularly interspaced short palindromic repeats (CRISPR)-encoded immunity in Type I systems relies on the Cascade (CRISPR-associated complex for antiviral defence) ribonucleoprotein complex, which triggers foreign DNA degradation by an accessory Cas3 protein. To establish the mechanism for adaptive immunity provided by the Streptococcus thermophilus CRISPR4-Cas (CRISPR-associated) system (St-CRISPR4-Cas), we isolated an effector complex (St-Cascade) containing 61-nucleotide CRISPR RNA (crRNA). We show that St-Cascade, guided by crRNA, binds in vitro to a matching proto-spacer if a proto-spacer adjacent motif (PAM) is present. Surprisingly, the PAM sequence determined from binding analysis is promiscuous and limited to a single nucleotide (A or T) immediately upstream (-1 position) of the proto-spacer. In the presence of a correct PAM, St-Cascade binding to the target DNA generates an R-loop that serves as a landing site for the Cas3 ATPase/nuclease. We show that Cas3 binding to the displaced strand in the R-loop triggers DNA cleavage, and if ATP is present, Cas3 further degrades DNA in a unidirectional manner. These findings establish a molecular basis for CRISPR immunity in St-CRISPR4-Cas and other Type I systems.

Clustered regularly interspaced short palindromic repeat (CRISPR) is a recently discovered adaptive prokaryotic immune system that provides acquired immunity against foreign nucleic acids by utilizing small guide crRNAs (CRISPR RNAs) to interfere with invading viruses and plasmids. In Escherichia coli, Cas3 is essential for crRNA-guided interference with virus proliferation. Cas3 contains N-terminal HD phosphohydrolase and C-terminal Superfamily 2 (SF2) helicase domains. Here, we provide the first report of the cloning, expression, purification and in vitro functional analysis of the Cas3 protein of the Streptococcus thermophilus CRISPR4 (Ecoli subtype) system. Cas3 possesses a single-stranded DNA (ssDNA)-stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes. Cas3 also shows ATP-independent nuclease activity located in the HD domain with a preference for ssDNA substrates. To dissect the contribution of individual domains, Cas3 separation-of-function mutants (ATPase(+)/nuclease(-) and ATPase(-)/nuclease(+)) were obtained by site-directed mutagenesis. We propose that the Cas3 ATPase/helicase domain acts as a motor protein, which assists delivery of the nuclease activity to Cascade-crRNA complex targeting foreign DNA.