Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease.

Cas9, the RNA-guided DNA endonuclease from the CRISPR-Cas (clustered regularly interspaced short palindromic repeat-CRISPR-associated) system, has been adapted for genome editing and gene regulation in multiple model organisms. Here we characterize a Cas9 ortholog from Streptococcus thermophilus LMG18311 (LMG18311 Cas9). In vitro reconstitution of this system confirms that LMG18311 Cas9 together with a trans-activating RNA (tracrRNA) and a CRISPR RNA (crRNA) cleaves double-stranded DNA with a specificity dictated by the sequence of the crRNA. Cleavage requires not only complementarity between crRNA and target but also the presence of a short motif called the PAM. Here we determine the sequence requirements of the PAM for LMG18311 Cas9. We also show that both the efficiency of DNA target cleavage and the location of the cleavage sites vary based on the position of the PAM sequence.

Targeted base editing in rice with CRISPR/ScCas9 system

The widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease derives its DNA targeting specificity from protein-DNA contacts with protospacer adjacent motif (PAM) sequences, in addition to base-pairing interactions between its guide RNA and target DNA. Previous reports have established that the PAM specificity of SpCas9 can be altered via positive selection procedures for directed evolution or other protein engineering strategies. Here we exploit in vivo directed evolution systems that incorporate simultaneous positive and negative selection to evolve SpCas9 variants with commensurate or improved activity on NAG PAMs relative to wild type and reduced activity on NGG PAMs, particularly YGG PAMs. We also show that the PAM preferences of available evolutionary intermediates effectively determine whether similar counterselection PAMs elicit different selection stringencies, and demonstrate that negative selection can be specifically increased in a yeast selection system through the fusion of compensatory zinc fingers to SpCas9.

The CRISPR-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA:DNA base-pairing to target foreign DNA in bacteria. Cas9:guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9:RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9:RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9:RNA. DNA strand separation and RNA:DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 employs PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate dsDNA scission.

COSMID: A Web-based Tool for Identifying and Validating CRISPR/Cas Off-target Sites.

Structure and Engineering of Francisella novicida Cas9

The RNA-guided Cas9 endonucleases have revolutionized gene editing and regulation, but their targeting scope is limited by the protospacer adjacent motif (PAM) requirement. The most extensively used SpCas9 from Streptococcus pyogenes recognizes the NGG PAM via an RxR PAM-binding motif within its PAM-interaction (PI) domain. To overcome the strict PAM requirement, we identified and characterized a Cas9 ortholog from Streptococcus equinus HC5 (SeHCas9) that shows high sequence identity with SpCas9 but harbors a different RxQ PAM-binding motif. Complete PAM profiling revealed that SeHCas9 recognized an NAG PAM and accommodated NKG and NAW PAMs. We investigated the PAM interaction mechanism by identifying the crucial role of R1336 within the RxQ motif in determining PAM specificity, as well as the essentiality of two conserved residues (R1152 and Q1229) across Cas9 orthologs bearing the RxQ motif for PAM recognition. Further protein engineering created two variants, SeHdCas9-Q1229R and SeHdCas9-RR, that showed robust repression across an NNG and NNN PAM range, respectively. Our work proposes a novel Cas9 PAM interaction mechanism and establishes PAM-free Cas9 variants for bacterial gene control with almost no targeting restriction.

Cpf1s, the RNA-guided nucleases of the class II clustered regularly interspaced short palindromic repeats system require a short motive called protospacer adjacent motif (PAM) to be present next to the targeted sequence for their activity. The TTTV PAM sequence of As- and LbCpf1 nucleases is relatively rare in the genome of higher eukaryotic organisms. Here, we show that two other Cpf1 nucleases, Fn- and MbCpf1, which have been reported to utilize a shorter, more frequently occurring PAM sequence (TTN) when tested in vitro, carry out efficient genome modification in mammalian cells. We found that all four Cpf1 nucleases showed similar activities and TTTV PAM preferences. Our approach also revealed that besides their activities their PAM preferences are also target dependent. To increase the number of the available targets for Fn- and MbCpf1 we generated their RVR and RR mutants with altered PAM specificity and compared them to the wild-type and analogous As- and LbCpf1 variants. The mutants gained new PAM specificities but retained their activity on targets with TTTV PAMs, redefining RR-Cpf1’s PAM-specificities as TTYV/TCCV, respectively. These variants may become versatile substitutes for wild-type Cpf1s by providing an expanded range of targets for genome engineering applications.

The proliferation of CRISPR/Cas9-based methods in Caenorhabditis elegans has enabled efficient genome editing and precise genomic tethering of Cas9 fusion proteins. Experimental designs using CRISPR/Cas9 are currently limited by the need for a protospacer adjacent motif (PAM) in the target with the sequence NGG. Here we report the characterization of two modified Cas9 proteins in C. elegans that recognize NGA and NGCG PAMs. We found that each variant could stimulate homologous recombination with a donor template at multiple loci and that PAM specificity was comparable to that of wild-type Cas9. To directly compare effectiveness, we used CRISPR/Cas9 genome editing to generate a set of assay strains with a common single-guide RNA (sgRNA) target sequence, but that differ in the juxtaposed PAM (NGG, NGA, or NGCG). In this controlled setting, we determined that the NGA PAM Cas9 variant can be as effective as wild-type Cas9. We similarly edited a genomic target to study the influence of the base following the NGA PAM. Using four strains with four NGAN PAMs differing only at the fourth position and adjacent to the same sgRNA target, we observed that efficient homologous replacement was attainable with any base in the fourth position, with an NGAG PAM being the most effective. In addition to demonstrating the utility of two Cas9 mutants in C. elegans and providing reagents that permit CRISPR/Cas9 experiments with fewer restrictions on potential targets, we established a means to benchmark the efficiency of different Cas9::PAM combinations that avoids variations owing to differences in the sgRNA sequence.

Crystal Structure of Staphylococcus aureus Cas9

CRISPR-Cas enzymes must recognize a protospacer-adjacent motif (PAM) to edit a genomic site, greatly limiting the range of targetable sequences in a genome. Although engineering strategies to alter PAM specificity exist, they typically require labor-intensive, iterative experimentation. We introduce an evolution-informed deep learning model, Protein2PAM, to efficiently guide the design of Cas protein variants tailored to recognize specific PAMs. Trained on a dataset of over 45,000 CRISPR-Cas PAMs, Protein2PAM rapidly and accurately predicts PAM specificity directly from Cas proteins across type I, II and V CRISPR-Cas systems. Using in silico mutagenesis, the model identifies residues critical for PAM recognition in Cas9 without using structural information. We use Protein2PAM to computationally evolve Nme1Cas9, generating variants with broadened PAM recognition and up to a 50-fold increase in PAM cleavage rates compared to the wild type in vitro. Our machine learning approach allows Cas enzymes to target sequences that were previously inaccessible because of PAM constraints, potentially increasing target flexibility in personalized genome editing.

Structural Plasticity of PAM Recognition by Engineered Variants of the RNA-Guided Endonuclease Cas9

Popular RNA-guided DNA endonuclease Cas9 from Streptococcus pyogenes (SpCas9) recognizes the canonical 5'-NGG-3' protospacer adjacent motif (PAM) and triggers double-stranded DNA cleavage activity. Mutations in SpCas9 were demonstrated to expand the PAM readability and hold promise for therapeutic and genome editing applications. However, the energetics of the PAM recognition and its relation to the atomic structure remain unknown. Using the X-ray structure (precatalytic SpCas9:sgRNA:dsDNA) as a template, we calculated the change in the PAM binding affinity in response to SpCas9 mutations using computer simulations. The E1219V mutation in SpCas9 fine-tunes the water accessibility in the PAM binding pocket and promotes new interactions in the SpCas9:noncanonical T-rich PAM, thus weakening the PAM stringency. The nucleotide-specific interaction of two arginine residues (i.e., R1333 and R1335 of SpCas9) ensured stringent 5'-NGG-3' PAM recognition. R1335A substitution (SpCas9R1335A) completely disrupts the direct interaction between SpCas9 and PAM sequences (canonical or noncanonical), accounting for the loss of editing activity. Interestingly, the double mutant (SpCas9R1335A,E1219V) boosts DNA binding affinity by favoring protein:PAM electrostatic contact in a desolvated pocket. The underlying thermodynamics explain the varied DNA cleavage activity of SpCas9 variants. A direct link between the energetics, structures, and activity is highlighted, which can aid in the rational design of improved SpCas9-based genome editing tools.

The CRISPR-Cas9 system locates targets through guide RNA pairing and recognition of a protospacer-adjacent motif (PAM). Although PAM specificity is sequence-determined, DNA topology can relax PAM requirements and enable near-PAMless cleavage by the type II-C Alicyclobacillus tengchongensis Cas9 (AtCas9). However, the structural mechanism underlying this regulation remains unknown. Here we report cryogenic-electron microscopy (cryo-EM) structures of AtCas9 bound to B-form DNA or a 340 bp underwound minicircle DNA containing wild-type or mutant PAMs. Despite PAM sequences differences, all three underwound complexes adopt an almost identical architecture distinct from the B-form DNA-bound state. On B-form DNA, AtCas9 recognizes the PAM through base-specific hydrogen bonds and steric exclusion, conferring preference for N4CNNN and N4RNNA (R = A/G). By contrast, underwound DNA widens the PAM major groove and promotes sequence-independent backbone contacts, explaining the near-PAMless cleavage. These findings uncover a topology-dependent mechanism of PAM recognition and establish a cryo-EM platform using underwound minicircle DNA for structural studies under native-like topological states.

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