Fig. 5: Minimal edits in phage T4 enabled by Cas13a counterselection.
From: Broad-spectrum CRISPR-Cas13a enables efficient phage genome editing
a, Homologous recombination vector design consists of a recoded Cas13a protospacer flanked by 52 bp of homology to the phage genome. b, Recoding design for a T4 non-essential gene, soc, with introduced silent mutations shown in magenta. Three designs with differing mutations were tested (soc-C, soc-S, soc-F). Underlined nucleotides represent the edge of the Cas13a CRISPR repeat. c, Recoding design for a T4 essential gene, dnap, with introduced silent mutations shown in magenta. Three designs with differing degrees of mutations were tested (dnap-C, dnap-S, dnap-F). Underlined nucleotides represent the edge of the Cas13a CRISPR repeat. d, Editing penetrance from three biological replicates of the editing and enrichment process shown in b for soc-C, soc-S and soc-F. Edited phage lysates with no detectable plaques are noted with ND. e, Editing penetrance from three biological replicates of the editing and enrichment process shown in b for dnap-C, dnap-S and dnap-F. Editing penetrance in d and e are presented as mean ± s.d. f, Unbiased sequencing of T4soc loci from individual plaques from three independent editing attempts. Deviations from wildtype are highlighted. g, Unbiased sequencing of T4dnap loci from individual plaques after editing attempts dnap-C (top), dnap-S (middle), and dnap-F (bottom), each with three independent editing attempts. Deviations from wildtype are highlighted. Sanger sequencing traces for all verified plaques including those shown in f and g can be found in Supplementary Figs. 16 and 20.
