Non-target DNA Strand Research Articles

Probing Electrostatic Interactions in DNA-Bound CRISPR/Cas9 Complexes by Molecular Dynamics Simulations.

Engineered protein mutations may be exploited to tune molecular interactions in the cellular environment. Here, we have explored the structural consequences of different Cas9 mutations in genome-editing CRISPR/Cas9 systems by means of Molecular Dynamics simulations. We have characterized mutation-induced structural changes and their implications for changes in protein-DNA, DNA-RNA, and DNA-DNA interactions. We present the analysis of multiple trajectories over the cumulative time scale of 7.7 μs, focusing on triple mutations that have been associated with enhancement of genome editing specificity, as well as control mutations. We find that the structural changes induced by the protein mutations are consistent with decreasing the strength of the interaction between Cas9 and the nontarget DNA strand. We discuss the implications of this finding for genome editing specificity.

Molecular mechanism of plasmid elimination by the DdmDE defense system.

Seventh-pandemic Vibrio cholerae strains contain two pathogenicity islands that encode the DNA defense modules DdmABC and DdmDE. In this study, we used cryogenic electron microscopy to determine the mechanistic basis for plasmid defense by DdmDE. The helicase-nuclease DdmD adopts an autoinhibited dimeric architecture. The prokaryotic Argonaute protein DdmE uses a DNA guide to target plasmid DNA. The structure of the DdmDE complex, validated by in vivo mutational studies, shows that DNA binding by DdmE triggers disassembly of the DdmD dimer and loading of monomeric DdmD onto the nontarget DNA strand. In vitro studies indicate that DdmD translocates in the 5'-to-3' direction, while partially degrading the plasmid DNA. These findings provide critical insights into the mechanism of DdmDE systems in plasmid elimination.

HLTF disrupts Cas9-DNA post-cleavage complexes to allow DNA break processing

The outcome of CRISPR-Cas-mediated genome modifications is dependent on DNA double-strand break (DSB) processing and repair pathway choice. Homology-directed repair (HDR) of protein-blocked DSBs requires DNA end resection that is initiated by the endonuclease activity of the MRE11 complex. Using reconstituted reactions, we show that Cas9 breaks are unexpectedly not directly resectable by the MRE11 complex. In contrast, breaks catalyzed by Cas12a are readily processed. Cas9, unlike Cas12a, bridges the broken ends, preventing DSB detection and processing by MRE11. We demonstrate that Cas9 must be dislocated after DNA cleavage to allow DNA end resection and repair. Using single molecule and bulk biochemical assays, we next find that the HLTF translocase directly removes Cas9 from broken ends, which allows DSB processing by DNA end resection or non-homologous end-joining machineries. Mechanistically, the activity of HLTF requires its HIRAN domain and the release of the 3′-end generated by the cleavage of the non-target DNA strand by the Cas9 RuvC domain. Consequently, HLTF removes the H840A but not the D10A Cas9 nickase. The removal of Cas9 H840A by HLTF explains the different cellular impact of the two Cas9 nickase variants in human cells, with potential implications for gene editing.

Exploiting activation and inactivation mechanisms in type I-C CRISPR-Cas3 for genome-editing applications

Type I CRISPR-Cas systems utilize the RNA-guided Cascade complex to identify matching DNA targets and the nuclease-helicase Cas3 to degrade them. Among the seven subtypes, type I-C is compact in size and highly active in creating large-sized genome deletions in human cells. Here, we use four cryoelectron microscopy snapshots to define its RNA-guided DNA binding and cleavage mechanisms in high resolution. The non-target DNA strand (NTS) is accommodated by I-C Cascade in a continuous binding groove along the juxtaposed Cas11 subunits. Binding of Cas3 further traps a flexible bulge in NTS, enabling NTS nicking. We identified two anti-CRISPR proteins AcrIC8 and AcrIC9 that strongly inhibit Neisseria lactamica I-C function. Structural analysis showed that AcrIC8 inhibits PAM recognition through allosteric inhibition, whereas AcrIC9 achieves so through direct competition. Both Acrs potently inhibit I-C-mediated genome editing and transcriptional modulation in human cells, providing the first off-switches for type I CRISPR eukaryotic genome engineering.

Mechanistic and evolutionary insights into a type V-M CRISPR-Cas effector enzyme.

RNA-guided type V CRISPR-Cas12 effectors provide adaptive immunity against mobile genetic elements (MGEs) in bacteria and archaea. Among diverse Cas12 enzymes, the recently identified Cas12m2 (CRISPR-Cas type V-M) is highly compact and has a unique RuvC active site. Although the non-canonical RuvC triad does not permit dsDNA cleavage, Cas12m2 still protects against invading MGEs through transcriptional silencing by strong DNA binding. However, the molecular mechanism of RNA-guided genome inactivation by Cas12m2 remains unknown. Here we report cryo-electron microscopy structures of two states of Cas12m2-CRISPR RNA (crRNA)-target DNA ternary complexes and the Cas12m2-crRNA binary complex, revealing structural dynamics during crRNA-target DNA heteroduplex formation. The structures indicate that the non-target DNA strand is tightly bound to a unique arginine-rich cluster in the recognition (REC) domains and the non-canonical active site in the RuvC domain, ensuring strong DNA-binding affinity of Cas12m2. Furthermore, a structural comparison of Cas12m2 with TnpB, a putative ancestor of Cas12 enzymes, suggests that the interaction of the characteristic coiled-coil REC2 insertion with the protospacer-adjacent motif-distal region of the heteroduplex is crucial for Cas12m2 to engage in adaptive immunity. Collectively, our findings improve mechanistic understanding of diverse type V CRISPR-Cas effectors and provide insights into the evolution of TnpB to Cas12 enzymes.

Twisting and swiveling domain motions in Cas9 to recognize target DNA duplexes, make double-strand breaks, and release cleaved duplexes.

The CRISPR-associated protein 9 (Cas9) has been engineered as a precise gene editing tool to make double-strand breaks. CRISPR-associated protein 9 binds the folded guide RNA (gRNA) that serves as a binding scaffold to guide it to the target DNA duplex via a RecA-like strand-displacement mechanism but without ATP binding or hydrolysis. The target search begins with the protospacer adjacent motif or PAM-interacting domain, recognizing it at the major groove of the duplex and melting its downstream duplex where an RNA-DNA heteroduplex is formed at nanomolar affinity. The rate-limiting step is the formation of an R-loop structure where the HNH domain inserts between the target heteroduplex and the displaced non-target DNA strand. Once the R-loop structure is formed, the non-target strand is rapidly cleaved by RuvC and ejected from the active site. This event is immediately followed by cleavage of the target DNA strand by the HNH domain and product release. Within CRISPR-associated protein 9, the HNH domain is inserted into the RuvC domain near the RuvC active site via two linker loops that provide allosteric communication between the two active sites. Due to the high flexibility of these loops and active sites, biophysical techniques have been instrumental in characterizing the dynamics and mechanism of the CRISPR-associated protein 9 nucleases, aiding structural studies in the visualization of the complete active sites and relevant linker structures. Here, we review biochemical, structural, and biophysical studies on the underlying mechanism with emphasis on how CRISPR-associated protein 9 selects the target DNA duplex and rejects non-target sequences.

Replacing the SpCas9 HNH domain by deaminases generates compact base editors with an alternative targeting scope

Base editors are RNA-guided deaminases that enable site-specific nucleotide transitions. The targeting scope of these Cas-deaminase fusion proteins critically depends on the availability of a protospacer adjacent motif (PAM) at the target locus and is limited to a window within the CRISPR-Cas R-loop, where single-stranded DNA (ssDNA) is accessible to the deaminase. Here, we reason that the Cas9-HNH nuclease domain sterically constrains ssDNA accessibility and demonstrate that omission of this domain expands the editing window. By exchanging the HNH nuclease domain with a monomeric or heterodimeric adenosine deaminase, we furthermore engineer adenine base editor variants (HNHx-ABEs) with PAM-proximally shifted editing windows. This work expands the targeting scope of base editors and provides base editor variants that are substantially smaller. It moreover informs of potential future directions in Cas9 protein engineering, where the HNH domain could be replaced by other enzymes that act on ssDNA.

Position of Deltaproteobacteria Cas12e nuclease cleavage sites depends on spacer length of guide RNA

ABSTRACT Cas12e proteins (formerly CasX) form a distinct subtype of Class II type V CRISPR-Cas effectors. Recently, it was shown that DpbCas12e from Deltaproteobacteria and PlmCas12e from Planctomycetes can introduce programmable double-stranded breaks in mammalian genomes. Thus, along with Cas9 and Cas12a Class II effectors, Cas12e could be harnessed for genome editing and engineering. The location of cleavage points in DNA targets is important for application of Cas nucleases in biotechnology. DpbCas12e was reported to produce extensive 5ʹ-overhangs at cleaved targets, which can make it superior for some applications. Here, we used high throughput sequencing to precisely map the DNA cut site positions of DpbCas12e on several DNA targets. In contrast to previous observations, our results demonstrate that DNA cleavage pattern of Cas12e is very similar to that of Cas12a: DpbCas12e predominantly cleaves DNA after nucleotide position 17–19 downstream of PAM in the non-target DNA strand, and after the 22nd position of target strand, producing 3–5 nucleotide-long 5ʹ-overhangs. We also show that reduction of spacer sgRNA sequence from 20nt to 16nt shifts Cas12e cleavage positions on the non-target DNA strand closer to the PAM, producing longer 6–8nt 5ʹ-overhangs. Overall, these findings advance the understanding of Cas12e endonucleases and may be useful for developing of DpbCas12e-based biotechnology instruments.

Engineering of CRISPR-Cas12b for human genome editing

The type-V CRISPR effector Cas12b (formerly known as C2c1) has been challenging to develop for genome editing in human cells, at least in part due to the high temperature requirement of the characterized family members. Here we explore the diversity of the Cas12b family and identify a promising candidate for human gene editing from Bacillus hisashii, BhCas12b. However, at 37 °C, wild-type BhCas12b preferentially nicks the non-target DNA strand instead of forming a double strand break, leading to lower editing efficiency. Using a combination of approaches, we identify gain-of-function mutations for BhCas12b that overcome this limitation. Mutant BhCas12b facilitates robust genome editing in human cell lines and ex vivo in primary human T cells, and exhibits greater specificity compared to S. pyogenes Cas9. This work establishes a third RNA-guided nuclease platform, in addition to Cas9 and Cpf1/Cas12a, for genome editing in human cells.

Mechanistic Insights into the cis- and trans-Acting DNase Activities of Cas12a

CRISPR-Cas12a (Cpf1) is an RNA-guided DNA-cutting nuclease that has been repurposed for genome editing. Upon target DNA binding, Cas12a cleaves both the target DNA in cis and non-target single-stranded DNAs (ssDNAs) in trans. To elucidate the molecular basis for both DNase cleavage modes, we performed structural and biochemical studies on Francisella novicida Cas12a. We show that guide RNA-target strand DNA hybridization conformationally activates Cas12a, triggering its trans-acting, non-specific, single-stranded DNase activity. In turn, cis cleavage of double-stranded DNA targets is a result of protospacer adjacent motif (PAM)-dependent DNA duplex unwinding, electrostatic stabilization of the displaced non-target DNA strand, and ordered sequential cleavage of the non-target and target DNA strands. Cas12a releases the PAM-distal DNA cleavage product and remains bound to the PAM-proximal DNA cleavage product in a catalytically competent, trans-active state. Together, these results provide a revised model for the molecular mechanisms of both the cis- and the trans-acting DNase activities of Cas12a enzymes, enabling their further exploitation as genome editing tools.

A PAM-Induced Signalling Activates the Communication between HNH and RUVC in CRISPR-Cas9

CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 is a bacterial immune system, which has revolutionized life sciences through the introduction of a facile genome editing technology. In this system, the endonuclease Cas9 associates with guide RNAs to match and cleave complementary DNA sequences, forming an RNA:DNA hybrid and a displaced non-target DNA strand. Thanks to a key DNA recognition element, a Protospacer Adjacent Motif (PAM), Cas9 performs site-specific DNA cleavages, enabling to engineer biological systems with unique efficiency and resulting in numerous applications in medicine and biotechnology. However, the mechanistic basis underlying the CRISPR-Cas9 function is unclear, preventing rational engineering of the system toward improved genome editing. We report on extensive molecular dynamics studies, including multi-microsecond and accelerated methods probing displacements over micro-to-milliseconds, characterizing the mechanism of conformational activation of Cas9, from its apo form up to the nucleic acid binding. These simulations disclosed a mechanism for RNA recruitment in which the domain relocations cause the formation of a positively charged cavity for nucleic acid binding. As well, we revealed the formation of a catalytically competent Cas9, which is prone for the catalysis of DNA. We showed how, upon DNA binding, the relocation of the catalytic HNH domain, assisted by conformational changes of the non-target DNA strand, activates Cas9 for catalysis. Finally, a mechanism of allosteric regulation, triggered by PAM binding and activating the catalytic domains for concerted catalysis has been discovered. Overall, our outcomes address the lack of mechanistic information on CRISPR-Cas9, improving our understanding of the activation process. This information is critical for structure-based design of the CRISPR-Cas9 system, which could impact the development of improved genome editing tools.

Duplex DNA Destabilization by Type V CRISPR-Cas Nucleases during Interrogation of DNA

The prokaryotic CRISPR-Cas effector complexes locate their target sites by scanning and interrogating the genomic DNA. The type II and type V CRISPR-Cas nucleases cleave double-stranded DNA bearing sequences complementary to guide segments of CRISPR RNAs (crRNA) flanked by conserved PAM sequences; they have been successfully used for genome editing in various organisms. Unpairing of a short PAM-proximal segment of DNA protospacer is a critical event that may determine the overall rate of target location. We sought to elucidate mechanistic details of initial DNA interrogation steps by type V CRISPR-Cas nucleases Cas12a and Cas12b (also known as Cpf1 and C2c1, respectively). Using fluorometric and biochemical assays, we studied interactions of Cas12a and Cas12b effectors with a set of model DNA substrates that mimic likely early intermediates on the pathway to the final R-loop complexes. Consistent with reported data on the mechanism of DNA interrogation by type II Cas9 nuclease, we find that binding of type V effectors to PAM favors separation of several adjacent to PAM DNA protospacer base pairs even in the absence of pairing between the crRNA guide and the protospacer. The separation is mediated in part by effector affinity for non-target DNA strand nucleotides in a single-stranded conformation. In contrast, effector interactions with the nearest to PAM target DNA strand nucleotides are energetically unfavorable in the type V early intermediate complexes, presumably due to a steric clash between the protein and DNA backbones. We suggest that these unfavorable interactions destabilize the DNA duplex and thus facilitate the DNA interrogation process. We discuss the similarities and differences between the mechanisms of DNA interrogation by Cas9, Cas12a and Cas12b effectors.

Efficient CRISPR/Cas9-Mediated Genome Editing Using a Chimeric Single-Guide RNA Molecule.

The CRISPR/Cas9 system has been applied in diverse eukaryotic organisms for targeted mutagenesis. However, targeted gene editing is inefficient and requires the simultaneous delivery of a DNA template for homology-directed repair (HDR). Here, we used CRISPR/Cas9 to generate targeted double-strand breaks and to deliver an RNA repair template for HDR in rice (Oryza sativa). We used chimeric single-guide RNA (cgRNA) molecules carrying both sequences for target site specificity (to generate the double-strand breaks) and repair template sequences (to direct HDR), flanked by regions of homology to the target. Gene editing was more efficient in rice protoplasts using repair templates complementary to the non-target DNA strand, rather than the target strand. We applied this cgRNA repair method to generate herbicide resistance in rice, which showed that this cgRNA repair method can be used for targeted gene editing in plants. Our findings will facilitate applications in functional genomics and targeted improvement of crop traits.

Structural Variation of Type I-F CRISPR RNA Guided DNA Surveillance

CRISPR-Cas systems are prokaryotic immune systems against invading nucleic acids. Type I CRISPR-Cas systems employ highly diverse, multi-subunit surveillance Cascade complexes that facilitate duplex formation between crRNA and complementary target DNA for R-loop formation, retention, and DNA degradation by the subsequently recruited nuclease Cas3. Typically, the large subunit recognizes bona fide targets through the PAM (protospacer adjacent motif), and the small subunit guides the non-target DNA strand. Here, we present the Apo- and target-DNA-bound structures of the I-Fv (type I-F variant) Cascade lacking the small and large subunits. Large and small subunits are functionally replaced by the 5' terminal crRNA cap Cas5fv and the backbone protein Cas7fv, respectively. Cas5fv facilitates PAM recognition from the DNA major groove site, in contrast to all other described type I systems. Comparison of the type I-Fv Cascade with an anti-CRISPR protein-bound I-F Cascade reveals that the type I-Fv structure differs substantially at known anti-CRISPR protein target sites and might therefore be resistant to viral Cascade interception.

Inhibition Mechanism of an Anti-CRISPR Suppressor AcrIIA4 Targeting SpyCas9

Prokaryotic CRISPR-Cas adaptive immune systems utilize sequence-specific RNA-guided endonucleasesto defend against infection by viruses, bacteriophages, and mobile elements, while these foreign genetic elements evolve diverse anti-CRISPR proteins to overcome the CRISPR-Cas-mediated defense of the host. Recently, AcrIIA2 and AcrIIA4, encoded by Listeria monocytogene prophages, were shown to block the endonuclease activity of type II-A Streptococcus pyogene Cas9 (SpyCas9). We now report the crystal structure of AcrIIA4 in complex with single-guide RNA-bound SpyCas9, thereby establishing that AcrIIA4 preferentially targets critical residues essential for PAM duplex recognition, as well as blocks target DNA access to key catalytic residues lining the RuvCpocket. These structural insights, validated bybiochemical assays on key mutants, demonstrate that AcrIIA4 competitively occupies both PAM-interacting and non-target DNA strand cleavage catalytic pockets. Our studies provide insights into anti-CRISPR-mediated suppression mechanisms for inactivating SpyCas9, thereby broadening the applicability of CRISPR-Cas regulatory tools for genome editing.

Structure Basis for Directional R-loop Formation and Substrate Handover Mechanisms in Type I CRISPR-Cas System

Type I CRISPR systems feature a sequential dsDNA target searching and degradation process, by crRNA-displaying Cascade and nuclease-helicase fusion enzyme Cas3, respectively. Here we present two cryo-EM snapshots of the Thermobifida fusca type I-E Cascade: (1) unwinding 11bp of dsDNA at the seed-sequence region to scout for sequence complementarity, and (2) further unwinding of the entire protospacer to form a full R-loop. These structures provide the much-needed temporal and spatial resolution to resolve key mechanistic steps leading to Cas3 recruitment. In the early steps, PAM recognition causes severe DNA bending, leading to spontaneous DNA unwinding to form a seed-bubble. The full R-loop formation triggers conformational changes in Cascade, licensing Cas3 to bind. The same process also generates a bulge in the non-target DNA strand, enabling its handover to Cas3 for cleavage. The combination of both negative and positive checkpoints ensures stringent yet efficient target degradation in type I CRISPR-Cas systems.

CRISPR-Cas9: Computational Insights Toward Improved Genome Editing

Life sciences are undergoing a transformative phase due to an emerging genome-editing technology based on the RNA-programmable CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9) system. In this system, the endonuclease Cas9 associates with a guide RNA to match and cleave complementary sequences in double stranded DNA, forming an RNA:DNA hybrid and a displaced non-target DNA strand. Although extensive structural studies are ongoing, the conformational dynamics of Cas9 and its interplay with the nucleic acids during association and DNA cleavage are largely unclear. This missing aspect hampers the precise structure-based design of CRISPR-Cas9 genome-editing tools with improved specificity. Here, we report the first biophysical study – based on extensive multi-microseconds molecular simulations integrated with structural data – revealing the conformational plasticity of Cas9 and identifying the key determinants that allow its large-scale conformational changes during nucleic acid binding and processing. We identify a remarkable conformational plasticity as an intrinsic property of the nuclease HNH domain, being a necessary factor allowing for the HNH domain repositioning during catalysis. More importantly, we disclose a key role of the non-target DNA during the process of activation of the HNH domain, showing how the non-target DNA positioning triggers local conformational changes that favor the formation of a catalytically competent Cas9. Our outcomes further suggest new and precise protein-engineering modifications, which are of fundamental importance for the rational design of more effective genome-editing tools. Overall, these novel findings constitute a reference for future experimental studies aimed at a full characterization of the dynamic features and at the improvement of biological applications of the CRISPR-Cas9 system.

Does Cas9-Catalyzed DNA Cleavage Generate Blunt Ends or Staggered Ends? Insight from Molecular Dynamic Simulations

The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (spCas9) along with a single guide RNA (sgRNA) has merged as a versatile toolbox for genome editing. Despite recent advances in the mechanistic studies on spCas9-sgRNA-mediated target double-stranded DNA (dsDNA) recognition and cleavage, it is still unclear how the catalytic Mg2+ ions induce the conformation changes toward the catalytic active state. It also remains controversial whether Cas9 generates blunt-ended or staggered-ended breaks with overhangs in the target DNA. To investigate these issues, here we performed the first all-atom molecular dynamics simulations of the spCas9-sgRNA-dsDNA system with and without Mg2+ bound. The simulation results show that binding of two Mg2+ ions at the RuvC domain active site could lead to structurally and energetically favorable coordination ready for the non-target DNA strand cleavage. Importantly, we argue that Cas9-catalyzed target DNA cleavage produces 1-bp staggered ends rather than generally assumed blunt ends.

Mechanism of Duplex DNA Destabilization by RNA-Guided Cas9 Nuclease during DNA Interrogation

The Cas9 endonuclease cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA (gRNA), which are flanked by a conserved PAM sequence. The Cas9-gRNA system has been successfully used for genome editing in various organisms. The Cas9-gRNA effector complex locates the target site by scanning and interrogating the genomic DNA. Unpairing of a short PAM-proximal segment of DNA is a critical event in DNA interrogation that may determine the overall rate of target location. We sought to elucidate mechanism of initial DNA interrogation steps that precede the pairing of target DNA with gRNA. Using fluorometric and biochemical assays, we studied Cas9-gRNA complexes with model DNA substrates likely mimicking early intermediates on the pathway to the final Cas9-gRNA-DNA R-loop complex. The results show that Cas9-gRNA binding to PAM favors separation of few nearest to PAM base pairs but causes no significant effect on DNA duplex stability in the protospacer region distant from PAM. The destabilization of DNA duplex is achieved primarily through interactions of Cas9-gRNA with non-target DNA strand nucleotides and hindering of duplex DNA entry into the Cas9-gRNA interior. The data also suggest that interaction of the target DNA strand with the “phosphate lock” loop of Cas9 is subsequent to the initial destabilization step and is coupled with the DNA-gRNA pairing.

Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations.

The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (spCas9) along with a single guide RNA (sgRNA) has emerged as a versatile toolbox for genome editing. Despite recent advances in the mechanism studies on spCas9-sgRNA-mediated double-stranded DNA (dsDNA) recognition and cleavage, it is still unclear how the catalytic Mg2+ ions induce the conformation changes toward the catalytic active state. It also remains controversial whether Cas9 generates blunt-ended or staggered-ended breaks with overhangs in the DNA. To investigate these issues, here we performed the first all-atom molecular dynamics simulations of the spCas9-sgRNA-dsDNA system with and without Mg2+ bound. The simulation results showed that binding of two Mg2+ ions at the RuvC domain active site could lead to structurally and energetically favorable coordination ready for the non-target DNA strand cleavage. Importantly, we demonstrated with our simulations that Cas9-catalyzed DNA cleavage produces 1-bp staggered ends rather than generally assumed blunt ends.