CRISPR from Prevotella and Francisella 1 (Cpf1) is an effector endonuclease of the class 2 CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) gene editing system. We developed a method for evaluating Cpf1 activity, based on target sequence composition in mammalian cells, in a high-throughput manner. A library of >11,000 target sequence and guide RNA pairs was delivered into human cells using lentiviral vectors. Subsequent delivery of Cpf1 into this cell library induced insertions and deletions (indels) at the integrated synthetic target sequences, which allowed en masse evaluation of Cpf1 activity by using deep sequencing. With this approach, we determined protospacer-adjacent motif sequences of two Cpf1 nucleases, one from Acidaminococcus sp. BV3L6 (hereafter referred to as AsCpf1) and the other from Lachnospiraceae bacterium ND2006 (hereafter referred to as LbCpf1). We also defined target-sequence-dependent activity profiles of AsCpf1, which enabled the development of a web tool that predicts the indel frequencies for given target sequences (http://big.hanyang.ac.kr/cindel). Both the Cpf1 characterization profile and the in vivo high-throughput evaluation method will greatly facilitate Cpf1-based genome editing.

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The authors would like to thank D.-S. Jang (Medical Illustrator, Medical Research Support Section, Yonsei University College of Medicine) for his help with the illustrations, D. Kim for developing Python programs, M. K. Song (Biostatistics Collaboration Unit, Yonsei University College of Medicine) and Y. Kim for the assistance with statistical analysis, E.-S. Lee for proofreading, R. Gopalappa for assistance with sample preparation, Severance Biomedical Science Institute for bioinformatics analysis, and B. Kleinstiver and K. Joung (both at Massachusetts General Hospital) for sharing raw data for Supplementary Figure 9 and Figure 3d. This work was supported in part by the National Research Foundation of Korea (grants 2014R1A1A1A05006189 (H.K.), 2013M3A9B4076544 (H.K.), 2014M3C9A3063541 (J.-W.N.), and 2015R1A2A1A15052668 (H.K.)), the Korean Health Technology R&D Project, Ministry of Health and Welfare, Republic of Korea (grants HI14C2019 (Medistar program) (H.K.), HI16C1012 (H.K.), and HI15C1578 (J.-W.N.)), and a faculty research grant of Yonsei University College of Medicine for 2015 (6-2015-0086 (H.K.)).

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Author notes

    • Hui K Kim
    •  & Myungjae Song

    These authors contributed equally to this work.


  1. Department of Pharmacology, Yonsei University College of Medicine, Seoul, South Korea.

    • Hui K Kim
    • , Myungjae Song
    • , Soobin Jung
    •  & Hyongbum Kim
  2. Brain Korea 21 Plus Project for Medical Sciences, Yonsei University College of Medicine, Seoul, South Korea.

    • Hui K Kim
    • , Soobin Jung
    •  & Hyongbum Kim
  3. Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, South Korea.

    • Myungjae Song
    •  & Hyun C Koh
  4. College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, South Korea.

    • Jinu Lee
  5. Department of Life Science, College of Natural Sciences, Hanyang University, Seoul, South Korea.

    • A Vipin Menon
    • , Young-Mook Kang
    •  & Jin-Wu Nam
  6. Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, South Korea.

    • Jae W Choi
    •  & Hyongbum Kim
  7. Drug Target Structure Research Center, Korea Institute of Bioscience and Biotechnology, Daejeon, South Korea.

    • Euijeon Woo
  8. Department of Pharmacology, College of Medicine, Hanyang University, Seoul, South Korea.

    • Hyun C Koh
  9. Graduate Program of Nano Science and Technology, Yonsei University, Seoul, South Korea.

    • Hyongbum Kim


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H.K.K. and M.S. performed most of the experiments (including library preparation, lentivirus generation, cell culture, and deep-sequencing experiments), analyzed the data, and contributed to the writing of the manuscript; J.L. generated lentivirus, determined MOI after transduction, and contributed to the writing of the manuscript; A.V.M., Y.-M.K., and J.-W.N. developed the computation model for AsCpf1 indel frequency prediction and contributed to the writing of the manuscript; S.J. significantly contributed to the performance of the experiments including cell culture, lentivirus generation, and deep-sequencing; J.W.C. calculated chromatin accessibility scores and performed related analyses; E.W. contributed to the analysis of target sequence nucleotide preferences based on the structure of AsCpf1 and to the writing of the manuscript; H.C.K. contributed to the writing of the manuscript; and H.K. conceived and supervised the study, analyzed the data, and wrote the manuscript.

Competing interests

Yonsei University has filed a patent based on this work, in which H.K.K., M.S., and H.K. are co-inventors.

Corresponding author

Correspondence to Hyongbum Kim.

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