Molecular basis for the PAM expansion and fidelity enhancement of an evolved Cas9 nuclease

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have been harnessed as powerful genome editing tools in diverse organisms. However, the off-target effects and the protospacer adjacent motif (PAM) compatibility restrict the therapeutic applications of these systems. Recently, a Streptococcus pyogenes Cas9 (SpCas9) variant, xCas9, was evolved to possess both broad PAM compatibility and high DNA fidelity. Through determination of multiple xCas9 structures, which are all in complex with single-guide RNA (sgRNA) and double-stranded DNA containing different PAM sequences (TGG, CGG, TGA, and TGC), we decipher the molecular mechanisms of the PAM expansion and fidelity enhancement of xCas9. xCas9 follows a unique two-mode PAM recognition mechanism. For non-NGG PAM recognition, xCas9 triggers a notable structural rearrangement in the DNA recognition domains and a rotation in the key PAM-interacting residue R1335; such mechanism has not been observed in the wild-type (WT) SpCas9. For NGG PAM recognition, xCas9 applies a strategy similar to WT SpCas9. Moreover, biochemical and cell-based genome editing experiments pinpointed the critical roles of the E1219V mutation for PAM expansion and the R324L, S409I, and M694I mutations for fidelity enhancement. The molecular-level characterizations of the xCas9 nuclease provide critical insights into the mechanisms of the PAM expansion and fidelity enhancement of xCas9 and could further facilitate the engineering of SpCas9 and other Cas9 orthologs.

Fig 1. In vitro and vivo activity of xCas9 compared to WT SpCas9.

(A) In vitro cleavage assays of xCas9 and WT SpCas9 using linearized plasmids containing a target sequence adjacent to TGG, TGA, TGT, and TGC PAMs. (B) Cytosine base editing efficiency of pBECKP and pBECKP-xCas9 plasmids toward target sequence adjacent to different PAMs in the K. pneumoniae strain KP_1.6366. The experiments were performed in triplicate, and one representative result was shown. The data underlying this figure can be found in S1 Data. (C) Cytosine base editing efficiency of pBECKP and pBECKP-xCas9 plasmids towards five different spacers in the K. pneumoniae strain KP_1.6366. Left panel: fully matched spacer; right panel: 18 and 19 mismatched spacer. The data underlying this figure can be found in S1 Data. PAM, protospacer adjacent motif; SpCas9, Streptococcus pyogenes Cas9; WT, wild-type.

Fig 2. Overall structures of xCas9.

(A) Domain organization of xCas9. The positions of seven amino acid substitutions are labeled with asterisk. (B) Cartoon representation showing the overall structure of the xCas9/sgRNA/DNA complex (TGG PAM). (C) Structural comparison of xCas9 (TGG PAM) and WT SpCas9 (PDB_ID: 4UN3). xCas9 and WT SpCas9 are colored cyan and white gray, respectively. CTD, C-terminal domain; HNH, HNH-like nuclease; NUC lobe, nuclease lobe; PAM, protospacer adjacent motif; REC lobe, α-helical recognition lobe; RuvC, RuvC-like nuclease; sgRNA, single-guide RNA; SpCas9, Streptococcus pyogenes Cas9; WT, wild-type.

Fig 3. Structural comparison of xCas9 with WT SpCas9.

(A) Overall structure superimposition of xCas9 and WT SpCas9 (PDB_ID: 4UN3). WT SpCas9 is colored white gray. Left panel: xCas9/TGC versus WT SpCas9; right panel: xCas9/TGA versus WT SpCas9. (B) Superimposition of the RNA/DNA heteroduplex-bound REC lobe of WT SpCas9 with that of xCas9 in complex with TGC (left) and TGA (right) PAMs. PAM, protospacer adjacent motif; REC lobe, α-helical recognition lobe; SpCas9, Streptococcus pyogenes Cas9; WT, wild-type.

Fig 4. Structural insight into PAM recognition by xCas9.

(A) Zoom-in views of PAM recognition by xCas9 in complex of TGC, TGA, TGG, and CGG PAMs, respectively. Upper panel: cartoon-view of PAM recognition in xCas9. Lower panel: The 2F_o-F_c electron density maps for the PAM nucleotides and V1219, R1333, and R1335 residues are contoured at 1.0 sigma level. (B) Superimposition of PAM recognition sites of the TGG PAM-bound SpCas9 with those of TGC, TGA, TGG, and CGG PAM-bound xCas9. PAM, protospacer adjacent motif; SpCas9, Streptococcus pyogenes Cas9.

Fig 5. Structural comparison of SpCas9/TGA and SpCas9/CGA with SpCas9/TGG.

(A) Superimposition of the overall structures of SpCas9/TGA and SpCas9/CGA with SpCas9/TGG (PDB_ID: 4UN3). (B) Superimposition of the REC lobe of SpCas9/TGG with those of SpCas9 in complex with TGA (left) and CGA (right) PAMs. (C) Close-up view of the PAM recognition sites of TGA and CGA PAM-bound SpCas9. Upper panel: cartoon-view of PAM recognition in SpCas9. Lower panel: The 2F_o-F_c electron density maps are contoured at 1.0 sigma level for the PAM nucleotides and V1219, R1333, and R1335. (D) Structural superimposition of PAM recognition sites of the TGG PAM-bound SpCas9 with those of TGA and CGA PAM-bound SpCas9. In (A) and (B), color codes of SpCas9/TGA and SpCas9/CGA are the same as those shown in Fig 1A, and SpCas9/TGG is colored in white gray. PAM, protospacer adjacent motif; REC lobe, α-helical recognition lobe; SpCas9, Streptococcus pyogenes Cas9; WT, wild-type.

Fig 6. In vitro and in vivo activity of WT SpCas9, xCas9, and xCas9 variants.

(A) In vitro cleavage assays of WT SpCas9, xCas9, and xCas9 variants using linearized plasmids containing a target sequence adjacent to TGG, TGA, TGT, and TGC PAMs. (B) In vitro cleavage assays of xCas9, xCas9V1219E, WT SpCas9, and SpCas9E1219V toward TGT targets. (C) Cytosine base editing efficiency of pBECKP, pBECKP-xCas9, and pBECKP-xCas9 variant plasmids in the K. pneumoniae strain KP_1.6366. Left panel: fully matched spacer; right panel: 18 and 19 mismatched spacer. The data underlying this figure can be found in S1 Data. PAM, protospacer adjacent motif; SpCas9, Streptococcus pyogenes Cas9; WT, wild-type.