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Updated: Feb 4, 2026

Genome Engineering of Primary Human B Cells Using CRISPR/Cas9
Published on: November 3, 2020
Engineering the Direct Repeat Sequence of crRNA for Optimization of FnCpf1-Mediated Genome Editing in Human Cells
Li Lin1, Xiubin He1, Tianyuan Zhao1
1School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang 325027, China; State Key Laboratory and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang 325027, China.
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.
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