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.
Highlights
The REC2 domain of xCas9 3.7 undergoes substantial structural rearrangement. It rotates away from the guide RNA/DNA heteroduplex and the REC1 domain to stabilize the conformation of REC3 domain (Fig. 1b)
Despite the conformational changes in REC2 and REC3 domains, they remain in contact with each other at the protospacer adjacent motif (PAM)-distal end, implying that these conformational changes in xCas9 3.7 may play an important role in enhancing DNA targeting specificity, which is discussed in detail below
DISCUSSION the stringent requirement for the PAM sequence enables the CRISPR-Cas immune system to efficiently distinguish ‘self’ from ‘non-self’, it restricts the selection of targetable sequences in the applications of genome editing
Summary
Most of archaea and many bacteria encode CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPRassociated proteins) adaptive immune systems to defend themselves from phage invasion.[1,2,3,4,5] In the type II CRISPR-Cas[9] system, the crRNA (CRISPR RNA):tracrRNA (trans-activating crRNA)-guided Cas[9] degrades double-stranded DNA (dsDNA) bearing a PAM (protospacer-adjacent motif) and an adjacent 20-nt sequence complementary to the crRNA.[6,7,8,9,10,11] The Cas[9] protein comprises a recognition (REC) lobe and a nuclease (NUC) lobe.[8,9,10] The REC lobe is composed of three REC domains (REC1-3) to recognize the guide RNA scaffold and guide RNA/DNA heteroduplex. The sgRNA (single-guide RNA)-guided S. pyogenes Cas[9] (SpCas9) system has been harnessed as the most widely used tool for genome manipulation, such as target gene disruption, transcriptional repression and activation, epigenetic modulation, and single base-pair conversion in various organisms and cell types.[12,13,14,15,16,17]
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