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

Nature Biomedical Engineering volume 1, Article number: 0066 (2017) | Download Citation


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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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.

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  1. Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, USA.

    • Bin Li
    • , Weiyu Zhao
    • , Xiao Luo
    • , Xinfu Zhang
    • , Chunxi Zeng
    •  & Yizhou Dong
  2. Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, Florida 32610, USA.

    • Chenglong Li
  3. Department of Biomedical Engineering, The Ohio State University, Columbus, Ohio 43210, USA.

    • Yizhou Dong
  4. The Center for Clinical and Translational Science, The Ohio State University, Columbus, Ohio 43210, USA.

    • Yizhou Dong
  5. James Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, USA.

    • Yizhou Dong
  6. Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio 43210, USA.

    • Yizhou Dong
  7. Department of Radiation Oncology, The Ohio State University, Columbus, Ohio 43210, USA.

    • Yizhou Dong


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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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Yizhou Dong.

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