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

Nature Biomedical Engineering volume 1, Article number: 0066 (2017) Cite this article

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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.

To increase the genome editing efficiency of CRISPR–Cas systems, previous studies have explored a variety of approaches16–19. For example, chemical modifications of CRISPR–Cas9 led to enhanced activity in a number of human cells18,19. Also, a chimaeric single-guide RNA with three chemically modified nucleotides at both the 5′ and 3′ ends strongly improved Cas9-mediated genome editing in human primary T cells18. And the incorporation of chemically modified nucleotides in guide RNAs has been shown to retain insertion and deletion (indel) percentages in the CRISPR–Cas9 nuclease system19. Furthermore, the structure of guide RNAs also plays a notable role in gene cutting for the CRISPR–Cas9 system20,21. Yet, to the best of our knowledge, the genome editing efficiency and off-target effects of engineered crRNAs and Cpf1 mRNAs have not been explored. Here, we report the systematic investigation of 42 chemically or structurally engineered crRNAs, and establish comprehensive structure–activity (genome editing efficiency) relationships. We also show that a ψ-modification is a suitable chemical alteration for AsCpf1 mRNA. Notably, we demonstrate that the combination of lead crRNA and ψ-mRNA substantially increased the gene-cutting efficiency by over 300% compared to the control group. This combination induced a more pronounced improvement in the gene-cutting efficiency when using LbCpf1 than when using AsCpf1. We also show that the applicability of crRNAs from Cpf1 orthologues is different for AsCpf1 and LbCpf1, as LbCpf1 is more conservative in the recognition of the loop structure at the 5′ handle. Our findings could facilitate a wide 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.

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Authors and Affiliations

  1. Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, The Ohio State University, Columbus, 43210, Ohio, 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, 32610, Florida, USA

    Chenglong Li

  3. Department of Biomedical Engineering,

    Yizhou Dong

  4. The Ohio State University, Columbus, 43210, Ohio, USA

    Yizhou Dong

  5. The Center for Clinical and Translational Science,

    Yizhou Dong

  6. The Ohio State University, Columbus, 43210, Ohio, USA

    Yizhou Dong

  7. James Comprehensive Cancer Center, The Ohio State University, Columbus, 43210, Ohio, USA

    Yizhou Dong

  8. Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, Columbus, 43210, Ohio, USA

    Yizhou Dong

  9. Department of Radiation Oncology, The Ohio State University, Columbus, 43210, Ohio, USA

    Yizhou Dong

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  1. Bin Li
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Contributions

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.

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Correspondence to Yizhou Dong.

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The authors declare no competing financial interests.

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Li, B., Zhao, W., Luo, X. et al. Engineering CRISPR–Cpf1 crRNAs and mRNAs to maximize genome editing efficiency. Nat Biomed Eng 1, 0066 (2017). https://doi.org/10.1038/s41551-017-0066

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