In vivo high-throughput profiling of CRISPR-Cpf1 activity

Title
In vivo high-throughput profiling of CRISPR-Cpf1 activity
Author
남진우
Issue Date
2017-01
Publisher
NATURE PUBLISHING GROUP
Citation
NATURE METHODS, v. 14, no. 2, page. 153-159
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 Cpfl 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 LbCpfl). 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.
URI
https://www.nature.com/articles/nmeth.4104http://repository.hanyang.ac.kr/handle/20.500.11754/106118
ISSN
1548-7091; 1548-7105
DOI
10.1038/NMETH.4104
Appears in Collections:
COLLEGE OF NATURAL SCIENCES[S](자연과학대학) > LIFE SCIENCE(생명과학과) > Articles
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