Structural basis of Faecalibaculum rodentium Cas9 with high genome editing efficiency and fidelity provide insights into CRISPR-Cas protein engineering

Bacteria Faecalibaculum rodentium (Fr) CRISPR-Cas9 system exhibits efficient cleavage activity and high genome editing fidelity by recognizing the 5'-NNTA-3' protospacer adjacent motif (PAM) sequence. This system may have an advantage in targeting the TATA box within eukaryotic promoters. However, the structural information of FrCas9 is largely unknown. Herein, we report two cryo-electron microscopy structures of FrCas9:sgRNA:DNA complex capturing the R-loop expansion and pre-catalytic status, at 2.89 angstrom and 3.6 angstrom resolution respectively. These complex structures elucidate the unique recognition patterns of the 5'-NRTA-3' PAM and sgRNA:DNA heteroduplex, highlighting specific key residues in FrCas9 nuclease activity and fidelity. We confirmed that the phosphate lock loop region of FrCas9 protein potentially enhances the tugging effects on the target-strand phosphate between the PAM and PAM-proximal +1 base pair, facilitating R-loop nucleation through the introduction of linear positively charged side chain residue substitutions. The genome-wide unbiased identification of DNA double-stranded breaks enabled by sequencing (GUIDE-seq) assays results show that FrCas9 phosphate lock loop variant V1103K improved the overall fidelity on 9 gene targets by up to 32.25%. Combined with alternations of heteroduplex interacting residues, a series of structural-based designed FrCas9 variants display synergistic effects on editing efficiency and fidelity in human cells. Together, our experiments provide the most detailed structural information for FrCas9 to date, and present the mechanism framework of a high fidelity Cas9 member with potential broadly applications.