Fig. 4: 2xN peptides expressed independently of Cas9 or Cas9-2xN fusions do not compensate for boxB-proximal subPAM interactions and can limit TraCT.
From: Engineered transcription-associated Cas9 targeting in eukaryotic cells
a Simplified schematic illustration similar to Fig. 1d, except that a Cas9-2xN fusion is shown co-expressed with an mCherry−2xN (mC−2xN) fusion in the same strain; either can interact with a nascent boxB RNA hairpin. b Transformation-associated editing fractions measured for Cas9MT3−2xN or native Cas9MT3 in SSA reporter strains with one boxB HT upstream of their protospacer and mC−2xN fusion proteins expressed in trans at the indicated levels (+/++/+++), or not at all (–). Measurements were performed as in Fig. 1f; data shown for the strain lacking mC−2xN (–) are replotted from Fig. 1f (1x boxB, MT3) to facilitate comparisons. Error bars, mean ± s.d. (n = 3, biological replicates). p values were calculated using unpaired, two-tailed t tests with Welch’s correction, Gaussian distributions assumed, and no correction for multiple comparisons. Key elements used to create images in this figure were adapted from “Engineered dual selection for directed evolution of SpCas9 PAM specificity.” Nat Commun 12, 349 (2021) by Goldberg, G. W. et al., under CC BY 4.0. Source data are provided as a Source Data file.
