Fig. 1

NmeCas9 expression and activity in human (HEK293T) cells. a Western blot detection of HA-tagged NmeCas9 in transiently transfected HEK293T cells. Lane 1: cells transfected with SpyCas9 plasmid under the control of the CMV promoter. Lane 2: cells transfected with NmeCas9 plasmid under the control of the elongation factor-1α (EF1α) promoter. Lane 3: cells expressing NmeCas9 and a non-targeting sgRNA (nt-sgRNA), which lacks a complementary site in the human genome. Lane 4: cells expressing NmeCas9 and a sgRNA targeting chromosomal site NTS3. Upper panel: anti-HA western blot. Lower panel: anti-GAPDH western blot as a loading control. b NmeCas9 targeting co-transfected split-GFP reporter with ps9, ps24, and ps25 sites. Plasmid cleavage by SpyCas9 is used as a positive control, and a reporter without a guide-complementary site (No ps: no protospacer) is used as a negative control to define background levels of recombination leading to GFP+ cells. c NmeCas9 programmed independently with different sgRNAs targeting eleven genomic sites flanked by an N₄GATT PAM, detected by T7E1 analysis. Products resulting from NmeCas9 genome editing are denoted by the red dots. d Quantitation of editing efficiencies from three independent biological replicates performed on different days. Error bars indicate ± standard error of the mean (± s.e.m.). e Editing efficiencies for chromosomal target sites as measured by PCR and high-throughput sequencing (Deep sequencing). Data are mean values ± s.e.m. from three biological replicates performed on different days. f Genomic edits with NmeCas9 programmed independently with different guides in different cell lines and using different methods of delivery