Fig. 2: Cleavage-dependent selections in S. cerevisiae converge on a doubly substituted PID variant with altered PAM preference.

a Schematic summary of the single-strand annealing (SSA) reporters employed for dual selection in yeast. The endogenous LYS2 and CAN1 marker loci are disrupted with identical protospacer insertions (PS1 or PS2, with PAMs in green or red) flanked by stop codons and ~100 bp direct repeat sequences which are not naturally duplicated in their respective native loci. Transformation-associated assembly of the Cas9 backbone with a complementing PID fragment allows for cleavage-dependent reconstitution of one or both markers through the SSA pathway. A single SV40 nuclear localization signal (NLS) is fused to the C-terminus of Cas9. b Validation of the yeast system using two selection strains configured with CGG or GCC PAMs at their LYS2 and CAN1 loci as indicated. Strains were transformed with backbone DNA and either a Wt PID insert or no PID (Ø), and plated in parallel as 10-fold serial dilutions on positive- and dual-selection media, as well as control media to enumerate total transformants. c WebLogo³⁷ summaries of NNS-randomized residues identified in non-Wt isolates from the positive-selection (top) or dual-selection (bottom) screening experiments. The number of isolates used to generate each plot is denoted below. Recoded Wt isolates were not detected in either of these experiments. d GFP activation assays comparing the KG variant with Wt, normalized as in Fig. 1b. PAMs present at the upstream protospacer in single-target reporter plasmids are indicated below. Error bars, mean ± s.d. (n = 3, biological replicates). *p = 0.0180; **p = 0.0099. Source data are available in the Source Data file.