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In vivo high-throughput profiling of CRISPR-Cpf1 activity

Authors
 Hui K Kim  ;  Myungjae Song  ;  Jinu Lee  ;  A Vipin Menon  ;  Soobin Jung  ;  Young-Mook Kang  ;  Jae W Choi  ;  Euijeon Woo  ;  Hyun C Koh  ;  Jin-Wu Nam  ;  Hyongbum Kim 
Citation
 NATURE METHODS, Vol.14 : 153-159, 2017 
Journal Title
NATURE METHODS
ISSN
 1548-7091 
Issue Date
2017
MeSH
Acidaminococcus/genetics ; Bacterial Proteins/genetics ; Bacterial Proteins/metabolism ; Clostridiales/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats* ; Endonucleases/genetics* ; Endonucleases/metabolism ; Francisella/genetics ; High-Throughput Screening Assays/methods* ; Humans ; Prevotella/genetics ; RNA, Guide* ; Transduction, Genetic
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.
Full Text
https://www.nature.com/nmeth/journal/v14/n2/full/nmeth.4104.html
DOI
10.1038/nmeth.4104
Appears in Collections:
1. College of Medicine (의과대학) > Dept. of Pharmacology (약리학교실) > 1. Journal Papers
Yonsei Authors
Kim, Hyongbum(김형범) ORCID logo https://orcid.org/0000-0002-4693-738X
URI
https://ir.ymlib.yonsei.ac.kr/handle/22282913/154339
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