Figure 1: CjCas9 and its PAM specificity.
From: In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni
(a) Cas9 orthologues and their sgRNAs. sgRNA structures were obtained using the mfold web server. See also Supplementary Fig. 1. (b) Schematic showing a PAM assay for characterization of PAM sequences recognized by Cas9. PCR amplicons containing a Cas9 target sequence (yellow) followed by randomized 10-bp (N10) sequences (green) were cleaved by CjCas9 in vitro. The sequence logo showing the PAM specificity was obtained using deep-sequencing data. (c) PAM specificity in cells. Surrogate reporters encoding GFP and RFP and containing a CjCas9 target sequence with degenerate PAMs were transfected with CjCas9 and its sgRNA plasmid into HEK 293 cells. The percentage of cells that express both GFP and RFP was normalized with that obtained using the optimal reporter containing the 5’-NNNNACAC-3’ PAM sequence. (−) represents a mock control. Error bars indicate s.e.m. (n=3).
