Engineered Cpf1 variants with altered PAM specificities

The utility of the Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) endonucleases is limited by their requirement of a TTTV protospacer adjacent motif (PAM) in the DNA substrate. To address this limitation, we engineered two AsCpf1 variants carrying the mutations S542R/K607R and S542R/K548V/N552R, which recognize TYCV and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Introducing these mutations at their corresponding positions in LbCpf1 similarly altered its PAM specificity. These variants increase the targeting range of Cpf1 by three-fold in human coding sequences to one cleavage site for every ~11 bp.