Fig. 1: Cas9 activity can be controlled by conditional interactions between auxiliary fusion domains and protospacer-proximal cis elements.
From: Engineered transcription-associated Cas9 targeting in eukaryotic cells
a General schematic of the LYS2 genetic reporter system, engineered with a heterologous protospacer (PS) insertion between 100 bp direct repeats. Co-expression of Cas9 with a targeting sgRNA licenses cleavage and deletion of the repeat-intervening sequences through SSA-based recombination that restores LYS2+. The native pLYS2 promoter (bent arrow) and an NGG PAM are used throughout the work, except where stated otherwise. b Transformation-associated editing with the Cas9-scTetR fusion or native Cas9 control (Ø) in yeast containing a tetO2 element 13 bp downstream of the PS. The edited fraction of LYS2+ transformants (out of total transformants) was measured for Wt, MT3, and NG PID variants using synthetic dropout plates (see “Methods”) that lack (–) or contain (+) Dox. Inset schematic illustrates Dox-induced dissociation of the scTetR domain. Error bars, mean ± s.d. (n = 6 or n = 3, biological replicates). c Same as in b but with Cas9-scRevTetR fusions as indicated (V10 or V16). Inset schematic illustrates Dox-induced binding of the scRevTetR domain. d Simplified schematic illustration of the Cas9-scMCP fusion interacting with a nascent MS2U/C RNA hairpin, generated from a single Hairpin Template (HT) upstream of the PS. The scMCP domain can be replaced with a 2xN domain that in principle allows binding of two boxB hairpins simultaneously. The nascent transcript is elongated at its 3’ end by an RNA polymerase (RNAP) transcribing left to right. e Transformation-associated editing fractions measured for MT3 and NG variants of Cas9-scMCP or native Cas9 in strains containing the indicated HTs. 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. f Same experimental setup as in e but performed with the 2xN domain in place of scMCP and different HT strains as indicated. 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.
