St1Cas9 requires a relatively strict PAM sequence (5'-NNRGAA-3', where R is A or G) for DNA targeting , significantly restricting the editing scope of CTBEWT and CGBEWT. We previously engineered a St1Cas9 variant KLKL by introducing D939K/E1057L/N1081K/K1086L mutations to relieve the PAM specificity . In wildtype St1Cas9, Q1084 and K1086 form bidentate hydrogen bond with the third A and fourth G when 5'-NNAGGA-3' PAM is used, and Q1084 is further stabilized by E1057 via a hydrogen bond (Fig. ). Mutations of E1057L and K1086L would disrupt basespecific interactions, while mutations of D939K and N1081K would introduce nonbase specific interactions . We recently identified that mutation of L1057Q in the KLKL variant would increase the in vitro DNA cleavage activity towards 5'-NNTTAA-3' PAM-and 5'-NNCTAA-3' PAM-containing DNAs (Fig. ). We therefore engineered a St1Cas9 KQKL variant containing D939K/E1057Q/N1081K/K1086L mutations to further relieve the PAM specificity. Comprehensive in vitro DNA cleavage assay revealed that mutation of D939K/E1057Q/N1081K/K1086L substantially expanded PAM recognition scope of the KQKL variant with the 5'-NNNNAA-3' PAM specificity compared with the 5'-NNRGAA-3' specificity of the wild-type St1Cas9 (Fig. ).