Figure 2.

Activity and Fidelity of the Loop Mutant

(A) Inactivation of GFP with point mutation in the Loop sequence. The guide sequence is shown in Figure 1B. (B) Inactivation of GFP using DR mutants. Sequences for guides are shown in (C)–(E). (C–E) Schematic of FnCpf1 crRNAs at sites 1 (C), 2 (D), and 3 (E) targeting GFP. Single-nucleotide mismatches are marked in red. (F) FnCpf1-mediated inactivation of GFP using FnCpf1 plus crRNA harboring DR mutants or DR wild-type with guides containing single mismatches at different positions. Inactivation of GFP is measured by FCM (flow cytometry). Relative activity (ratio) was determined by the formula (A − C)/(B − C) × 100%, where A, B, and C represent the activity of the mutant DR, wild-type (WT) DR, and negative control, respectively. As to the fidelity test (F), ratio was determined by the formula (A′ − C)/(B′ − C) × 100%, where A′, B′, and C represent the activity of the mismatched spacer, totally matched spacer, and negative control, respectively. Error bars, SEM; n = 3. *p < 0.05; **p < 0.01; ***p < 0.001. WT, wild-type.