Fig. 5.

Applications of the U-rich crRNA to multiplexed genome editing and PAM-divergent AsCpf1 variants. a Simultaneous improvements of the indel efficiencies of multiple targets by an array of U-rich crRNAs. crRNA-encoding sequences with three different targets and one scrambled one were cloned into the 3′-UTR region of the eGFP gene in the pEGFP-C1 vector (Clontech). The U-rich crRNAs have target sequences with a 20-base match plus an additional T4AT6, while each control crRNA only has 23-base match target sequences. HEK-293T cells were transfected with 5 μg each of Cpf1-encoding and crRNA-encoding vectors. The indel efficiency was calculated after normalization with the transfection efficiency as assessed by the green-fluorescent cell counts. b, c Application of U-rich crRNA to AsCpf1 PAM variants. *p < 0.001, **p < 0.01 (n = 3), two-tailed Student’s t test. b Three targets were selected as common targets for the WT and RR variant of AsCpf1, which have TTTA and TYCC PAM sequence in each strand. The indel efficiency of WT AsCpf1 or the RR variant was investigated in the presence of the canonical or U-rich crRNA. *p < 0.001, **p < 0.01 (n = 3), two-tailed Student’s t test. c Three different targets with a TTTA PAM sequence were subjected to indel mutation by transfecting HEK-293T cells with the WT or the RVR variant of AsCpf1, each of which was guided by either canonical or U-rich crRNA. *p < 0.001, (n = 3), two-tailed Student’s t test