title: In vivo high-throughput profiling of CRISPR-Cpf1 activity.
abstract: 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.
authors.affiliation: Department of Pharmacology, Yonsei University College of Medicine, Seoul, South Korea.|Brain Korea 21 Plus Project for Medical Sciences, Yonsei University College of Medicine, Seoul, South Korea.
authors.forename: Hui K
authors.initials: HK
authors.lastname: Kim
authors.affiliation: Department of Pharmacology, Yonsei University College of Medicine, Seoul, South Korea.|Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, South Korea.
authors.forename: Myungjae
authors.initials: M
authors.lastname: Song
authors.affiliation: College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, South Korea.
authors.forename: Jinu
authors.initials: J
authors.lastname: Lee
authors.affiliation: Department of Life Science, College of Natural Sciences, Hanyang University, Seoul, South Korea.
authors.forename: A Vipin
authors.identifier: 0000-0003-3603-909X
authors.initials: AV
authors.lastname: Menon
authors.affiliation: Department of Pharmacology, Yonsei University College of Medicine, Seoul, South Korea.|Brain Korea 21 Plus Project for Medical Sciences, Yonsei University College of Medicine, Seoul, South Korea.
authors.forename: Soobin
authors.initials: S
authors.lastname: Jung
authors.affiliation: Department of Life Science, College of Natural Sciences, Hanyang University, Seoul, South Korea.
authors.forename: Young-Mook
authors.initials: YM
authors.lastname: Kang
authors.affiliation: Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, South Korea.
authors.forename: Jae W
authors.initials: JW
authors.lastname: Choi
authors.affiliation: Drug Target Structure Research Center, Korea Institute of Bioscience and Biotechnology, Daejeon, South Korea.
authors.forename: Euijeon
authors.initials: E
authors.lastname: Woo
authors.affiliation: Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, South Korea.|Department of Pharmacology, College of Medicine, Hanyang University, Seoul, South Korea.
authors.forename: Hyun C
authors.initials: HC
authors.lastname: Koh
authors.affiliation: Department of Life Science, College of Natural Sciences, Hanyang University, Seoul, South Korea.
authors.forename: Jin-Wu
authors.identifier: 0000-0003-0047-3687
authors.initials: JW
authors.lastname: Nam
authors.affiliation: Department of Pharmacology, Yonsei University College of Medicine, Seoul, South Korea.|Brain Korea 21 Plus Project for Medical Sciences, Yonsei University College of Medicine, Seoul, South Korea.|Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, South Korea.|Graduate Program of Nano Science and Technology, Yonsei University, Seoul, South Korea.
authors.forename: Hyongbum
authors.identifier: 0000-0002-4693-738X
authors.initials: H
authors.lastname: Kim
mesh_terms: D045850:Acidaminococcus / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D000076987:CRISPR-Associated Protein 9; D000068016:Eubacteriales / Q000235:genetics; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D005603:Francisella / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D018720:Prevotella / Q000235:genetics; D000094704:RNA, Guide, CRISPR-Cas Systems; D014161:Transduction, Genetic
chemical_list: D001426:Bacterial Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases