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. Author manuscript; available in PMC: 2018 Aug 28.
Published in final edited form as: Nature. 2018 Feb 28;556(7699):57–63. doi: 10.1038/nature26155

Figure 1. Phage-assisted continuous evolution (PACE) of Cas9 variants with broadened PAM compatibility.

Figure 1

a, PACE takes place in a fixed-volume “lagoon” that is continuously diluted with fresh host E. coli cells. Upon infection, each selection phage (SP) that encodes a Cas9 variant capable of binding the target PAM and protospacer on the accessory plasmid (AP) induces expression of gene III, resulting in infectious progeny phage that propagate the active Cas9 variant in subsequent host cells. b, Anatomy of a phage-infected host cell during PACE. The host cell carries the AP, which links Cas9 target DNA binding to phage propagation, and the mutagenesis plasmid (MP), which elevates mutagenesis during PACE. c, d, The crystal structure of SpCas9 with the location of xCas9 mutations shown. e, Genotypes of some evolved xCas9 variants, colored by evolution stage. See Supplementary Table 5 for 95 xCas9 variant genotypes.