Extended Data Figure 1. Optimization of Cas9 PACE.

Luciferase expression in E. coli was used as a proxy of gene III expression during efforts to link Cas9 binding to gene expression for PACE. a, b, Seven guide RNAs targeting the luciferase reporter (G1–G7′, see Supplementary Table 1), as well as a scrambled guide RNA negative control (G0) were tested without dCas9 (white bars) and with ω–dCas9 (a) or dCas9–ω (b) fusions (grey bars). c, Tests of seven different linkers between ω and dCas9. See Supplementary Table 2 for linker sequences. d, Evolution of ω–dCas9 on an NGG PAM site in PACE yielded variants (PACE1, PACE2, and PACE3) that were tested in comparison with canonical (“wt”) ω–dCas9, ω tethered to PACE1 dCas9, and the I12N ω mutant tethered to canonical dCas9. Values and error bars reflect the mean and s.d. of n=3 biologically independent samples.