한빛사 논문
Abstract
Hui K Kim1,2,10, Myungjae Song1,3,10, Jinu Lee4, A Vipin Menon5, Soobin Jung1,2, Young-Mook Kang5, Jae W Choi6, Euijeon Woo7, Hyun C Koh3,8, Jin-Wu Nam5 & Hyongbum Kim1,2,6,9,*
1Department of Pharmacology, Yonsei University College of Medicine, Seoul, South Korea. 2Brain Korea 21 Plus Project for Medical Sciences, Yonsei University College of Medicine, Seoul, South Korea. 3Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, South Korea. 4College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, South Korea. 5Department of Life Science, College of Natural Sciences, Hanyang University, Seoul, South Korea. 6Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, South Korea. 7Drug Target Structure Research Center, Korea Institute of Bioscience and Biotechnology, Daejeon, South Korea. 8Department of Pharmacology, College of Medicine, Hanyang University, Seoul, South Korea. 9Graduate Program of Nano Science and Technology, Yonsei University, Seoul, South Korea. 10These authors contributed equally to this work.
*Correspondence to : Hyongbum Kim
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.
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