Nature. 2018 Apr 5;556(7699):57-63. doi:10.1038/nature26155

Hu JH, Miller SM, Geurts MH, Tang W, Chen L, Sun N, Zeina CM, Gao X, Rees HA, Lin Z, Liu DR.

Evolved Cas9 variants with broad PAM compatibility and high DNA specificity.

Screened using phage-assisted continuous evolution (PACE) in an assay where a catalytically inactive Cas9 protein fused to the ω subunit of bacterial RNA polymerase activates the promoter of an essential phage gene allowing for the reproduction of the phage and the transmission of advantageous mutations in the Cas protein. The phage gene is provided through a library of constructs differing in their protospacer adjacent motif (PAM).