Engineering the Direct Repeat Sequence of crRNA for Optimization of FnCpf1-Mediated Genome Editing in Human Cells

Abstract

FnCpf1-mediated genome-editing technologies have enabled a broad range of research and medical applications. Recently, we reported that FnCpf1 possesses activity in human cells and recognizes a more compatible PAM (protospacer adjacent motif, 5'-KYTV-3'), compared with the other two commonly used Cpf1 enzymes (AsCpf1 and LbCpf1), which requires a 5'-TTTN-3' PAM. However, due to the efficiency and fidelity, FnCpf1-based clinical and basic applications remain a challenge. The direct repeat (DR) sequence is one of the key elements for FnCpf1-mediated genome editing. In principle, its engineering should influence the corresponding genome-editing activity and fidelity. Here we showed that the DR mutants [G(-9)A and U(-7)A] could modulate FnCpf1 performance in human cells, enabling enhancement of both genome-editing efficiency and fidelity. These newly identified features will facilitate the design and optimization of CRISPR-Cpf1-based genome-editing strategies.

Keywords: FnCpf1; direct repeat; genome editing.