Engineering CRISPR–Cpf1 crRNAs and mRNAs to maximize genome editing efficiency

Bin Li, Weiyu Zhao, Xiao Luo, Xinfu Zhang, Chenglong Li, Chunxi Zeng, Yizhou Dong

Index: 10.1038/s41551-017-0066

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Abstract

Cpf1, a type-V CRISPR–Cas effector endonuclease, exhibits gene-editing activity in human cells through a single RNA-guided approach. Here, we report the design and assessment of an array of 42 types of engineered Acidaminococcus sp. Cpf1 (AsCpf1) CRISPR RNA (crRNA) and 5 types of AsCpf1 mRNA in human cell lines. We show that the top-performing modified crRNA (cr3′5F, containing five 2′-fluoro ribose at the 3′ terminus) and AsCpf1 mRNA (full ψ-modification) improved gene-cutting efficiency by, respectively, 127% and 177%, with respect to unmodified crRNA and plasmid-encoding AsCpf1. We also show that the combination of cr3′5F and ψ-modified AsCpf1 or Lachnospiraceae bacterium Cpf1 (LbCpf1) mRNA augmented gene-cutting efficiency by over 300% with respect to the same control, and discovered that 11 out of 16 crRNAs from Cpf1 orthologues enabled genome editing in the presence of AsCpf1. Engineered CRISPR–Cpf1 systems should facilitate a broad range of genome editing applications.