Extended Data Figure 4. Directed evolution of wtGFP to EGFP using CRISPR-X.

a) A replicate of the wtGFP evolution experiment (Fig. 2a) was performed using electroporated sgRNAs and MS2-AIDΔ. Flow cytometry scatter plots are shown for the wtGFP parent and samples before each round of sorting. The wtGFP locus was sequenced for the unsorted condition and after both sorting rounds. Enrichment of mutation was calculated at each base position. The graphs of enrichment are shown for both wtGFP targeted and safe harbor targeted libraries except after Sort #2 where no safe harbor cells were recovered after sorting. Identified mutations are labeled in the graphs. b) wtGFP cells expressing dCas9, MS2-AIDΔ, and wtGFP were lentivirally infected with sgwtGFP.1 or sgSafe.2 in replicate and sorted once, enriching for spectrum-shifted GFP cells. Scatter plots for the parent and unsorted populations are shown for both replicates. The wtGFP locus was sequenced pre- and post-sorting, and enrichment of mutations at each base position is shown. The S65T mutation is labeled in the graph for the sorted condition.