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Genome-wide specificity of dCpf1 cytidine base editors

Author

Listed:
  • Daesik Kim

    (Genome Editing Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB)
    Center for Genome Engineering, Institute for Basic Science (IBS))

  • Kayeong Lim

    (Center for Genome Engineering, Institute for Basic Science (IBS)
    Seoul National University)

  • Da-eun Kim

    (Center for Genome Engineering, Institute for Basic Science (IBS)
    Seoul National University)

  • Jin-Soo Kim

    (Center for Genome Engineering, Institute for Basic Science (IBS)
    Seoul National University)

Abstract

Cpf1-linked base editors broaden the targeting scope of programmable cytidine deaminases by recognizing thymidine-rich protospacer-adjacent motifs (PAM) without inducing DNA double-strand breaks (DSBs). Here we present an unbiased in vitro method for identifying genome-wide off-target sites of Cpf1 base editors via whole genome sequencing. First, we treat human genomic DNA with dLbCpf1-BE ribonucleoprotein (RNP) complexes, which convert C-to-U at on-target and off-target sites and, then, with a mixture of E. coli uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endonuclease VIII, which removes uracil and produces single-strand breaks (SSBs) in vitro. Whole-genome sequencing of the resulting digested genome (Digenome-seq) reveals that, on average, dLbCpf1-BE induces 12 SSBs in vitro per crRNA in the human genome. Off-target sites with an editing frequency as low as 0.1% are successfully identified by this modified Digenome-seq method, demonstrating its high sensitivity. dLbCpf1-BEs and LbCpf1 nucleases often recognize different off-target sites, calling for independent analysis of each tool.

Suggested Citation

  • Daesik Kim & Kayeong Lim & Da-eun Kim & Jin-Soo Kim, 2020. "Genome-wide specificity of dCpf1 cytidine base editors," Nature Communications, Nature, vol. 11(1), pages 1-8, December.
    • Handle: RePEc:nat:natcom:v:11:y:2020:i:1:d:10.1038_s41467-020-17889-9
    DOI: 10.1038/s41467-020-17889-9
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