Structural basis for the promiscuous PAM recognition by Corynebacterium diphtheriae Cas9

Abstract

The RNA-guided DNA endonuclease Cas9 cleaves double-stranded DNA targets bearing a protospacer adjacent motif (PAM) and complementarity to an RNA guide. Unlike other Cas9 orthologs, Corynebacterium diphtheriae Cas9 (CdCas9) recognizes the promiscuous NNRHHHY PAM. However, the CdCas9-mediated PAM recognition mechanism remains unknown. Here, we report the crystal structure of CdCas9 in complex with the guide RNA and its target DNA at 2.9 Å resolution. The structure reveals that CdCas9 recognizes the NNRHHHY PAM via a combination of van der Waals interactions and base-specific hydrogen bonds. Moreover, we find that CdCas9 exhibits robust DNA cleavage activity with the optimal 22-nucleotide length guide RNAs. Our findings highlight the mechanistic diversity of the PAM recognition by Cas9 orthologs, and provide a basis for the further engineering of the CRISPR-Cas9 genome-editor nucleases.

Fig. 1

In vitro cleavage activities of Corynebacterium diphtheriae Cas9 (CdCas9). a In vitro cleavage activities of CdCas9 with the 20–24-nt guide single-guide RNAs (sgRNAs). The linearized plasmid target bearing the GGGAAAC protospacer adjacent motif (PAM) was incubated with the CdCas9-sgRNA complex at 37 °C for 0.5, 1, 2, and 5 min, and the cleavage products were then analyzed by a MultiNA microchip electrophoresis system. b In vitro cleavage activities of CdCas9 and Streptococcus pyogenes Cas9 (SpCas9). CdCas9 and SpCas9 were programmed with their 22- and 20-nt guide sgRNAs, respectively. The linearized plasmid target bearing the GGGAAAC PAM was incubated with the Cas9-sgRNA complex at 37 °C for 0.25, 0.5, 1, 2, and 5 min. c Motif obtained from the in vitro PAM identification assay. d PAM preference of CdCas9. The linearized plasmid targets bearing the different PAMs were incubated with CdCas9-sgRNA22 at 37 °C for 0.5 and 5 min. Error bars represent s.d. from n = 3 replicates

Fig. 2

Structure of the CdCas9-sgRNA-target DNA complex. a Domain structure of CdCas9. The HNH nuclease domain was truncated for crystallization. BH, bridge helix; PLL, phosphate-lock loop. b Schematics of the single-guide RNA (sgRNA) and the target DNA. The disordered region is enclosed in a dashed box. TS, target strand; NTS, non-target strand; SL1, stem loop 1; SL2, stem loop 2. c Overall structure of CdCas9-∆HNH in complex with the sgRNA and its target DNA. The BH is colored green. The disordered region of the sgRNA is shown as a dotted line. CdCas9, Corynebacterium diphtheriae Cas9; R:AR, repeat:anti-repeat duplex

Fig. 3

Structural comparison of the Cas9 orthologs. The structures of Corynebacterium diphtheriae Cas9 (CdCas9), Campylobacter jejuni Cas9 (CjCas9) (PDB: 5X2G), Staphylococcus aureus Cas9 (SaCas9) (PDB: 5CZZ), Streptococcus pyogenes Cas9 (SpCas9) (PDB: 4UN3), and Francisella novicida Cas9 (FnCas9) (PDB: 5B2O) bound to their cognate single-guide RNA (sgRNAs) and target DNAs, and the structure of Actinomyces naeslundii Cas9 (AnCas9) in the apo form (PDB: 4OGE). The bridge helix and the linker regions are colored green and light green, respectively

Fig. 4

Schematic of the nucleic acid recognition by Corynebacterium diphtheriae Cas9 (CdCas9). The residues that interact with the single-guide RNA (sgRNA) and its target DNA via their main chains are shown within parentheses. The disordered region is enclosed in a dashed box

Fig. 5

Structure and recognition of the single-guide RNA (sgRNA) scaffold. a Structure of the sgRNA scaffold. The guide region is omitted for clarity. The disordered region is shown as a dotted line. b Binding of the sgRNA scaffold to Corynebacterium diphtheriae Cas9 (CdCas9) (stereo view). The disordered region of the sgRNA is shown as a dotted line. c Effects of the sgRNA truncation. The linearized plasmid target with the GGGAAAC protospacer adjacent motif (PAM) was incubated at 37 °C for 0.5 and 5 min with CdCas9 and the truncated CdCas9 sgRNA (22-nt guide) or the Streptococcus pyogenes Cas9 (SpCas9) sgRNA (22-nt guide). 1–112, the full-length sgRNA; ∆57–65, the sgRNA, in which nucleotides 57–65 were replaced with GAAA; ∆87–107, the sgRNA, in which nucleotides 87–107 were replaced with GAAA; 1–81, the sgRNA, in which nucleotides 82–112 were truncated; SpCas9, the SpCas9 sgRNA. d Base-specific recognition of the sgRNA linker region (stereo view). Hydrogen bonds are shown as dashed lines. e Superimposition of the CdCas9 sgRNA and the SpCas9 sgRNA (PDB: 4OO8). The disordered region is shown as a dotted line. Error bars represent s.d. from n = 3 replicates

Fig. 6

Protospacer adjacent motif (PAM) recognition by Corynebacterium diphtheriae Cas9 (CdCas9). a Binding of the PAM duplex to CdCas9. b Schematics of the PAM recognition by CdCas9. Hydrogen bonds and hydrophobic interactions are depicted by black and red dashed lines, respectively. c Recognition of the GGGTAAT PAM (stereo view). Hydrogen bonds are shown as dashed lines. d Effects of the mutations on the PAM-interacting residues. The linearized plasmid target with the GGGAAAC PAM was incubated with the wild-type or the mutants of CdCas9, together with the single-guide RNA (sgRNA22), at 37 °C for 0.5 and 5 min. FPL, F1011A/P1043A/L1046A; FPLK, F1011A/K1015A/P1043A/L1046A. e Functional importance of the second PAM nucleotide. The linearized plasmid targets bearing the different PAMs were incubated with CdCas9-sgRNA22 at 37 °C for 0.5 and 5 min. Error bars represent s.d. from n = 3 replicates

Fig. 7

Protospacer adjacent motif (PAM) recognition by the Cas9 orthologs. a Structure-based sequence alignment of the PAM-interacting regions of the Cas9 orthologs. b–f PAM recognition by Streptococcus pyogenes Cas9 (SpCas9) (PDB: 4UN3) (b), Staphylococcus aureus Cas9 (SaCas9) (PDB: 5CZZ) (c), Corynebacterium diphtheriae Cas9 (CdCas9) (d), Campylobacter jejuni Cas9 (CjCas9) (PDB: 5X2G) (e), and Francisella novicida Cas9 (FnCas9) (PDB: 5B2O) (f). In SpCas9, the insertion between the β6 and β7 is omitted for clarity. The PAMs are highlighted in purple. Hydrogen bonds are shown as dashed lines. Water molecules are depicted as red spheres