Fig. 6

NmeCas9 guide length requirements in mammalian cells. a Split-GFP activity profile of NmeCas9 cleavage with ps9 sgRNAs bearing spacers of varying lengths (18–24 nt) along with 5′-terminal G residues to enable transcription. Bars represent mean values ± s.e.m. from three independent biological replicates performed on different days. b T7EI analysis of editing efficiencies at the NTS33 genomic target site (with an N₄GATT PAM) with sgRNAs bearing spacers of varying lengths (13–25 nt) with 1–2 5′-terminal G residues. Products resulting from NmeCas9 genome editing are denoted by the red dots. c Quantitation of editing efficiencies (of experiment in b) from three independent biological replicates performed on different days. Error bars indicate ± standard error of the mean (± s.e.m.). d As in (b), but targeting the NTS32 genomic site (with a N₄GCTT PAM). e Quantitation of editing efficiencies (of experiment in d) from three independent biological replicates performed on different days. Error bars indicate ± standard error of the mean (± s.e.m.)