Structural insights into a high fidelity variant of SpCas9

Abstract

The RNA-guided endonucleases of the CRISPR-Cas9 system, including the most widely used Cas9 from Streptococcus pyogenes (SpCas9), are becoming a robust genome editing tool in model organisms and hold immense promise for therapeutic applications. Many strategies have been employed to overcome the limitations caused by SpCas9’s off-target effects and its stringent requirement for the protospacer adjacent motif (PAM) sequence. However, the structural mechanisms underlying these strategies remain undefined. Here, we present crystal structure of a SpCas9 variant, xCas9 3.7 that has broad PAM compatibility and high DNA targeting specificity, in complex with a single-guide RNA and its double-stranded DNA targets. Structural comparison revealed that salt bridge-stabilized R1335 is critical for the stringent selection of PAM sequence by SpCas9. Unrestricted rotamerization of this residue by the E1219V mutation in xCas9 3.7 lessens the stringency for PAM recognition and allows SpCas9 to recognize multiple PAM sequences as further supported by biochemical data. Compared to those in wild-type (WT) SpCas9, REC2 and REC3 domains in xCas9 3.7 undergo striking conformational changes, leading to reduced contact with DNA substrate. SpCas9 mutants engineered to display less interaction with DNA and have conformationally more flexible REC2 and REC3 domains display enhanced specificity for DNA substrates in both biochemical and cellular assays. Taken together, our findings reveal the structural mechanisms underlying the broadened PAM compatibility and high DNA fidelity of xCas9 3.7, which can assist rational engineering of more efficient SpCas9 variants and probably other Cas9 orthologs.

Robust Genome Editing of Single-Base PAM Targets with Engineered ScCas9 Variants

“…xCas9-3.7 and SpCas9-NG additionally edit few sites that harbor 5'-CGN-3' and 5'-TGN-3' sequences, but perform poorly on all tested 5'-NGC-3' PAM targets, consistent with previously reported data (19,20,(22)(23)(24). Sc+ and Sc++, on the other hand, improve greatly upon the editing capabilities of the wild-type ScCas9 enzyme, demonstrating nearly 3-fold improvement in indel formation efficiency on certain 5'-NNGC-3' targets, and even editing sites at which ScCas9, xCas9-3.7, and SpCas9-NG have negligible activity ( Figure 2A).…”

“…SpCas9-NG and xCas9-3.7 both harbor various substitutions in their open reading frames (ORFs) that allow reduced specificity from the canonical 5'-NGG-3' to the more minimal 5'-NGN-3' PAM. Specifically, positions 1218-1219 for both enzymes have been shown to be the most consequential in terms of PAM recognition (20,24). To engineer ScCas9 to possess improved PAM targeting capabilities, we performed global pairwise alignments using the BLOSUM62 scoring matrix (25) of various Streptococcus Cas9 orthologs to SpCas9, xCas9-3.7, and SpCas9-NG at these critical residues.…”

Continuous evolution of SpCas9 variants compatible with non-G PAMs

“…As broadening the PAM targeting capabilities of some Cas9 variants has been shown to increase the proportion of genomic off-targets edits 5 , 7 , we characterized the DNA specificity of our variants by performing GUIDE-seq 37 on SpCas9, SpCas9-NRRH, SpCas9-NRCH, and SpCas9-NRTH in U2OS cells on four characterized on-target genomic sites 6 , 7 , 8 , 37 . For comparison, we also analyzed xCas9, which has greatly reduced off-target activity compared to SpCas9 6 , 38 – 40 . On all four sites examined, our evolved variants displayed higher on-target activity and similar or fewer numbers of detected off-target sites compared to SpCas9, though xCas9 was the most highly specific variant tested ( Fig.…”

“…2b , d – h ). Residue E1219 forms hydrogen bonds with R1335 in SpCas9, and mutations at this position are thought to destabilize the interaction between R1335 and the third PAM base 38 . Mutations at D1135 have been previously reported 7 , 8 and may modulate interactions with the sugar-phosphate backbone of the non-target DNA strand; R1114G and Q1221H could alter similar interactions.…”

Strategies for Developing CRISPR‐Based Gene Editing Methods in Bacteria

“…Thus, there is an urgent need for new engineered Cas nuclease which has a broadened editing range. Recently, a SpCas9 variant, xCas9, has been developed, which recognizes a PAM site as 5′‐NG‐3′ with increased fidelity . We envision that xCas9 may take the place of the canonical SpCas9.…”