Figure 4. SauCas9 repression of a GFP reporter in vivo.

(A) Comparison of dSpy and dSauCas9 to repress GFP expression on the DNA and RNA level. GFP signal is normalized to OD₆₀₀ to control for difference in cell density between samples. GFP/OD₆₀₀ ratios for guide alone and RNP are normalized to values for a non-targeting guide vector and an Apo protein control, respectively. Target sites were chosen to be adjacent to PAM sites for Spy, Sau, both, or neither as indicated. Note: the slight GFP repression observed with dSpyCas9 using the target sequence adjacent to the Sau PAM (CAGAGT) likely results from the ability of SpyCas9 to use an NAG PAM, albeit with reduced efficiency (Hsu et al., 2013). ****p<0.0001 by one-way ANOVA. (B) Relative expression of GFP using guides with different length targeting sequences. Target site here is the GFP2 sequence chosen for its robust targeting activity. (C) (Upper) Diagram of targeting sequences across the GFP mRNA and ribosome-binding site (RBS). (Lower) Relative expression of GFP of SauCas9 RNP normalized to sgRNA alone for targeting sequences across the GFP reporter. Dashed red line indicates that the sgRNA alone is as efficient as the RNP for GFP repression. (A–C) Bars represent mean ± S.D. (n = 3). (D) Model for observed SauCas9 ssRNA targeting activity. We propose that accessible RNA is cleaved or repressed efficiently while structured and protein-bound RNA is not targeted by SauCas9.

Figure 4—figure supplement 1. Repression of GFP mRNA.

(A) dSauCas9-sgRNA directed against a GFP mRNA (coding) or antisense (noncoding) sequence. GFP2 and GFP6 refer to guides diagrammed in Figure 4C. Dashed red line indicates that the sgRNA alone is as efficient as the SauCas9 RNP for GFP repression. Bars represent mean ± S.D. (n = 3). ***p<0.001, ****p<0.0001, by one-way ANOVA. (B) Electrophoretic mobility shift assay (EMSA) confirming that dSauCas9 does not bind dsDNA adjacent to non-canonical PAMs. Targeting sequence is identical for all substrates but with varied PAM sequences as indicated for the guides in Figure 4C. Final concentrations of dSauCas9 from left to right: 0, 0.1, 0.5, 1, 5, 10, 50, 100 nM. (C) Comparison of ability of dSau and wtSauCas9 to repress GFP expression in vivo. Bars represent mean ± S.D. (n = 3).