Engineering a Streptococcus Cas9 Ortholog with an RxQ PAM-Binding Motif for PAM-Free Gene Control in Bacteria

Figures

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  Figure 1. Characterization of SeHdCa9 with the RxQ PAM-binding motif. (A) Multiple sequence alignment of SpCas9-like orthologs bearing different PAM-binding motifs. (Red arrows indicate the residues selected for examination during mechanism investigation in Section 2.2). (B) The eGFP repression system: pZE12-luc harbors eGFP with randomized NNN inserted after the ATG start codon of eGFP and sgRNA, and pCS27 contains the catalytically deactivated SeHdCas9. The sgRNA was designed to adopt the NNN trinucleotides as a PAM. (C) Compete PAM profile of SeHdCas9 using the eGFP repression assay. (D) Impact of changing the fourth base following the NAG PAM on SeHdCas9 activity. All the in vivo tests were performed with three independent biological replicates.

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  Figure 2. Investigation of the PAM interaction mechanism of SeHCas9. (A) Impact of residue mutagenesis within the RxQ PAM-binding motif on SeHdCas9 activity. (B) eGFP repression efficiency of R1336- and Q1338-mutants on representative NNG and NAA PAMs. (C) Sequence alignment focusing on the critical residues R1152 and Q1229 that are highly conserved among orthologs bearing the RxQ motif. (Red arrows indicate the residues selected for examination during mechanism investigation). (D) Impact of PAM-proximal residue mutagenesis on SeHdCas9 activity. (E) Overlay of the structure model of the SeHCas9 PI domain (green) with the SpCas9 PI domain (light blue) in the crystal structure of SpCas9 (PDB ID: 4UN3, gray). The dsDNA is colored in yellow, and sgRNA is in orange. Zoom-in schematics illustrate the specific interactions (dotted line in dark gray) between the RxQ PAM-binding motif and NAG PAM in SeHCas9, in comparison with the interactions between the RxR motif and NGG PAM in SpCas9 (upper panel), and the contacts (dotted line in light gray) generated by R1152 and Q1229 in SeHCas9, in comparison with the contacts by their corresponding residues, S1136 and G1218, in SpCas9 (lower panel). (F)…

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  Figure 3. Structure-guided engineering of SeHdCas9 for PAM expansion. (A) Predicted structure of SeHCas9 with the engineering targets (colored in blue) shown. eGFP repression assay of SeHdCas9 variants with mutations on (B) W1337, (C) R1152, (D) Q1229, and (E) other residues against representative NNG and NAA PAMs. (F) Combinatorial mutations to further optimize the engineered variants. All the in vivo tests were performed with three independent biological replicates.

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  Figure 4. Complete PAM profile of the engineered SeHdCas9 variants, SeHdCas9-Q1229R and SeHdCas9-RR, in comparison with the SeHdCas9-WT, SpdRY, and SpdNG-LWQT on, respectively, single-G, single-A, single-T, and single-C PAMs using the eGFP repression assay. All the in vivo tests were performed with three independent biological replicates. Each dot represents one of the three biological replicates conducted in the repression test. ***P ≤ 0.001, ****P ≤ 0.0001 (two-tailed t-test).