Engineered SpCas9s and AsCas12a cleave fewer off-target genomic sites than wild-type (wt) Cas9. However, understanding their fidelity, mechanisms and cleavage outcomes requires systematic profiling ...across mispaired target DNAs. Here we describe NucleaSeq-nuclease digestion and deep sequencing-a massively parallel platform that measures the cleavage kinetics and time-resolved cleavage products for over 10,000 targets containing mismatches, insertions and deletions relative to the guide RNA. Combining cleavage rates and binding specificities on the same target libraries, we benchmarked five SpCas9 variants and AsCas12a. A biophysical model built from these data sets revealed mechanistic insights into off-target cleavage. Engineered Cas9s, especially Cas9-HF1, dramatically increased cleavage specificity but not binding specificity compared to wtCas9. Surprisingly, AsCas12a cleavage specificity differed little from that of wtCas9. Initial DNA cleavage sites and end trimming varied by nuclease, guide RNA and the positions of mispaired nucleotides. More broadly, NucleaSeq enables rapid, quantitative and systematic comparisons of specificity and cleavage outcomes across engineered and natural nucleases.
Prokaryotic CRISPR-Cas adaptive immune systems utilize sequence-specific RNA-guided endonucleases to 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 RuvC pocket. These structural insights, validated by biochemical 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.
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•Crystal structure of S. pyogenes Cas9 in complex with sgRNA and suppressor AcrIIA4•Selective recognition of pre-target bound SpyCas9 binary complex by AcrIIA4•Competitive binding of AcrIIA4 over dsDNA for SpyCas9-sgRNA binary complex•Mechanistic insights into blockage of SpyCas9 preventing dsDNA cleavage by AcrIIA4
Anti-CRISPRs are phage factors that inhibit bacterial CRISPR systems. Yang and Patel describe the structure of an anti-CRISPR protein, AcrIIA4, in complex with Cas9 with single-guide RNA. The structure reveals the mechanism of inhibition and provides the basis for developing tools to regulate Cas9 function in genome editing.
CRISPR–Cas9 represents a promising platform for genome editing, yet means for its safe and efficient delivery remain to be fully realized. A novel vehicle that simultaneously delivers the Cas9 ...protein and single guide RNA (sgRNA) is based on DNA nanoclews, yarn‐like DNA nanoparticles that are synthesized by rolling circle amplification. The biologically inspired vehicles were efficiently loaded with Cas9/sgRNA complexes and delivered the complexes to the nuclei of human cells, thus enabling targeted gene disruption while maintaining cell viability. Editing was most efficient when the DNA nanoclew sequence and the sgRNA guide sequence were partially complementary, offering a design rule for enhancing delivery. Overall, this strategy provides a versatile method that could be adapted for delivering other DNA‐binding proteins or functional nucleic acids.
All rolled into one: A biologically inspired delivery vehicle for CRISPR–Cas9 is based on yarn‐like DNA nanoparticles that are synthesized by rolling circle amplification. The DNA nanoclews were efficiently loaded with Cas9 protein/single guide RNA complexes and delivered them into human cells, enabling targeted gene disruption.
Bacterial class 2 CRISPR-Cas systems utilize a single RNA-guided protein effector to mitigate viral infection. We aggregated genomic data from multiple sources and constructed an expanded database of ...predicted class 2 CRISPR-Cas systems. A search for novel RNA-targeting systems identified subtype VI-D, encoding dual HEPN domain-containing Cas13d effectors and putative WYL-domain-containing accessory proteins (WYL1 and WYL-b1 through WYL-b5). The median size of Cas13d proteins is 190 to 300 aa smaller than that of Cas13a–Cas13c. Despite their small size, Cas13d orthologs from Eubacterium siraeum (Es) and Ruminococcus sp. (Rsp) are active in both CRISPR RNA processing and targeting, as well as collateral RNA cleavage, with no target-flanking sequence requirements. The RspWYL1 protein stimulates RNA cleavage by both EsCas13d and RspCas13d, demonstrating a common regulatory mechanism for divergent Cas13d orthologs. The small size, minimal targeting constraints, and modular regulation of Cas13d effectors further expands the CRISPR toolkit for RNA manipulation and detection.
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•Type VI-D is a CRISPR-Cas system with a Cas13d effector and a WYL domain accessory•Cas13d is an RNA-guided RNase approximately 20% smaller than Cas13a–Cas13c effectors•WYL1 positively modulates Cas13d target and collateral RNase activity•Cas13d has minimal sequence and secondary structure requirements for targeting
Compiling an expanded database of predicted class 2 CRISPR-Cas systems, Yan et al. identify and characterize subtype VI-D. Cas13d is an RNA-guided RNase effector with polyphyletic WYL-domain accessory proteins. One WYL1 ortholog enhances activity of divergent Cas13d orthologs. The small effector size and modular enhancement further expand RNA modification capabilities.
The CRISPR-associated protein Cas12a (Cpf1), which has been repurposed for genome editing, possesses two distinct nuclease activities: endoribonuclease activity for processing its own guide RNAs and ...RNA-guided DNase activity for target DNA cleavage. To elucidate the molecular basis of both activities, we determined crystal structures of Francisella novicida Cas12a bound to guide RNA and in complex with an R-loop formed by a non-cleavable guide RNA precursor and a full-length target DNA. Corroborated by biochemical experiments, these structures reveal the mechanisms of guide RNA processing and pre-ordering of the seed sequence in the guide RNA that primes Cas12a for target DNA binding. Furthermore, the R-loop complex structure reveals the strand displacement mechanism that facilitates guide-target hybridization and suggests a mechanism for double-stranded DNA cleavage involving a single active site. Together, these insights advance our mechanistic understanding of Cas12a enzymes and may contribute to further development of genome editing technologies.
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•Crystal structures of binary (crRNA) and ternary (R-loop) Cas12a complexes•Cas12a pre-orders the seed sequence of the crRNA to facilitate target binding•crRNA is processed via acid-base catalysis, generating a 2′,3′-cyclic phosphate•Cleavage of target and non-target DNA strands is catalyzed by the same active site
Swarts et al. determined crystal structures of Cas12a in complex with a guide RNA and in complex with both a guide RNA and a double-stranded target DNA, providing insights into the mechanisms of guide RNA processing, R-loop formation, and DNA cleavage.
CRISPR-Cas immune systems utilize RNA-guided nucleases to protect bacteria from bacteriophage infection. Bacteriophages have in turn evolved inhibitory “anti-CRISPR” (Acr) proteins, including six ...inhibitors (AcrIIA1–AcrIIA6) that can block DNA cutting and genome editing by type II-A CRISPR-Cas9 enzymes. We show here that AcrIIA2 and its more potent homolog, AcrIIA2b, prevent Cas9 binding to DNA by occluding protein residues required for DNA binding. Cryo-EM-determined structures of AcrIIA2 or AcrIIA2b bound to S. pyogenes Cas9 reveal a mode of competitive inhibition of DNA binding that is distinct from other known Acrs. Differences in the temperature dependence of Cas9 inhibition by AcrIIA2 and AcrIIA2b arise from differences in both inhibitor structure and the local inhibitor-binding environment on Cas9. These findings expand the natural toolbox for regulating CRISPR-Cas9 genome editing temporally, spatially, and conditionally.
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•Atomic cryo-EM structure of AcrIIA2- or AcrIIA2b-bound SpyCas9-sgRNA complex•AcrIIA2 inhibitor family shows a convergent Cas9 inhibition mechanism with AcrIIA4•AcrIIA2 exhibits a strong temperature-dependent anti-CRISPR activity
Jiang et al. report cryo-EM structures of type II-A anti-CRISPRs (AcrIIA2 and its homolog AcrIIA2b) bound to S. pyogenes Cas9, revealing a convergent inhibition mechanism between AcrIIA2 and AcrIIA4. The temperature-dependent differences between AcrIIA2 and AcrIIA2b provide an interesting condition-dependent variable that could be exploited for developing Cas9-based 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;
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.
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•Target ssDNA binding allosterically induces unblocking of the RuvC active site•PAM binding facilitates strand separation in dsDNA targets•Non-target DNA strand cleavage precedes target DNA strand cleavage•Cas12a releases the PAM-distal end of cleaved dsDNA targets
Swarts et al. elucidated two structures of Cas12a in complex with a guide RNA and with either single-stranded or double-stranded DNA targets. Together with biochemical assays, these structures reveal the mechanisms of Cas12a-mediated cis and trans cleavage of DNA substrates.
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