Figure 2: Optimization of sgRNA length for CjCas9.
From: In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni
(a) sgRNAs with variable lengths (19 to 23 nucleotide complementary with a target DNA sequence) were designed and transfected with CjCas9 plasmid into human HEK 293 cells. Genomic DNA was isolated 48 h after transfection. Indel frequencies were analysed by targeted deep sequencing. The first guanine nucleotide at the 5’ end that does not match the target sequence is shown in lower case. PAM motifs are shown in red. Error bars indicate s.e.m. (n=3). (b) Mutation frequencies at target sites in the mouse genome. Rosa26 and Tp53-specific gX22 guide RNAs were designed and transfected into mouse NIH 3T3 cells together with CjCas9 plasmid. Genome editing efficiencies were examined by deep sequencing using genome DNA isolated from cells after 48 h of transfection. PAM motifs are shown in red. Error bars indicate s.e.m. (n=3). See also Supplementary Fig. 2. (c) CjCas9-mediated genome editing at the human AAVS1 locus with different PAM sequences. sgRNAs targeting sites with a 5’-NNNNACAC-3’ PAM (green; 12 sgRNAs), 5′-NNNNATAC-3′ PAM (light blue; 7 sgRNAs), 5′-NNNNGCAC-3′ PAM (dark blue; 10 sgRNAs), and 5′-NNNNGTAC-3′ PAM (yellow; 8 sgRNAs) were designed and their activities examined in HEK293 cells with deep sequencing. Error bars indicate s.e.m. (n=3).
