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

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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.

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Figure 1: Design of chemically modified and structurally engineered crRNAs.
Figure 2: Gene-cutting efficiency of chemically modified and stem-engineered crRNAs.
Figure 3: Gene-cutting efficiency of chemically modified AsCpf1 mRNAs and loop-engineered crRNAs.
Figure 4: Maximizing the genome editing efficiency through the combination of chemically modified crRNA and AsCpf1 mRNA.
Figure 5: Engineered LbCpf1 crRNAs and mRNAs, and crRNA applicability.
Figure 6: Targeted deep-sequencing analysis of on-target and off-target gene cutting for chemically modified crRNA and AsCpf1 mRNA.
Figure 7: Targeted deep-sequencing analysis of on-target and off-target gene cutting for chemically modified Lbcr3′5F and LbCpf1 mRNA.

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Acknowledgements

We acknowledge F. Zhang and B. Zetsche for providing Cpf1 plasmids and technical assistance. This work was supported by the Early Career Investigator Award from the Bayer Hemophilia Awards Program, Research Awards from the National PKU Alliance, New Investigator Grant from the AAPS Foundation, R01HL136652 from the National Heart, Lung, and Blood Institute, as well as the start-up fund from the College of Pharmacy at The Ohio State University.

Author information

B.L. and Y.D. conceived and designed the experiments; B.L., W.Z., X.L., C.Z. and X.Z. performed the experiments; B.L., W.Z., X.L., X.Z., C.L., C.Z. and Y.D. analysed the data; B.L. and Y.D. wrote the paper with edits and comments from all authors.

Correspondence to Yizhou Dong.

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The authors declare no competing financial interests.

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