Extended Data Fig. 6: EvolvR-mediated mutagenesis can be coupled with a non-selectable genetic screen.

a, To test the capability for coupling EvolvR-mediated mutagenesis with a non-selectable genetic screen, we designed a target plasmid containing a GFP cassette with an early termination codon in the GFP coding sequence (pTarget-GFP*). After co-transforming pEvolvR with pTarget-GFP* and growing for 24 h, we analysed and sorted the GFP-positive fraction. In the two replicates expressing an off-target gRNA, we did not detect or sort any GFP cells. By contrast, for the two replicates expressing a gRNA nicking four nucleotides away from the chain-terminating mutation in the coding sequence of GFP, we found that 0.06% and 0.07% of the total cells were GFP positive. These results agree with sequencing outcomes from Fig. 1b, which showed that expressing nCas9–PolI3M for 24 h produces substitutions in the target region at frequencies between 0.5% to 1%. b, After culturing the sorted populations, both replicates expressing an off-target gRNA did not show growth, whereas both replicates expressing the on-target gRNA grew bright green.