Extended Data Fig. 5: ApoNmeCas9 exhibits acquisition-stimulatory activity and has distinct domain requirements as interference.
From: Cas9 senses CRISPR RNA abundance to regulate CRISPR spacer acquisition
a, Interference assay showing that a crRNA-tracrRNA pair or sgRNA is required for Cas9 to block transformation. Data are shown as in Extended Data Fig. 2k. b, Cas9 is expressed to comparable levels in strains used in Fig. 3. Top, anti-Cas9 western blot, leaky expression from uninduced Tet promoter; bottom, GroEL, loading control. c, NmeCas9 has two states: an apo- state that stimulates acquisition and an RNA-loaded state that ensures low acquisition. d, RNA EMSA showed that NmeCas9 engage cognate crRNA and tracrRNA as a pair in vitro. Binding was performed at 37 °C for 30 min, in the absence (“-”) or presence (“+”) of NmeCas9. e, NmeCas9 is protected from trypsin proteolysis by its crRNA-tracr pair. Purified apoCas9 was pre-incubated with various RNAs, trypsin digested for 5-, 15-, or 30- min, then analyzed by SDS-PAGE. Input, without trypsin. f, Schematic of strains used in Fig. 4. Blue box, cas9 deletion mutants complemented to genomic iga-trpB locus, with or without an sgRNA. g, All but one Cas9 mutant (ΔRuvCIΔBH) is expressed in Neisseria. Top, anti-Flag western blot to track Cas9; bottom, GroEL probed as a loading control. h, All domain deletion mutants tested exhibited major defects in CRISPR interference. Data are shown as in Extended Data Fig. 2k.
