In vivo high-throughput profiling of CRISPR-Cpf1 activity
- Title
- In vivo high-throughput profiling of CRISPR-Cpf1 activity
- Author
- 고현철
- Keywords
- NUCLEASE-INDUCED MUTATIONS; HUMAN-CELLS; GENOME; GUIDE; CPF1; RNA; SPECIFICITIES; ENRICHMENT; SYSTEM
- Issue Date
- 2017-02
- 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.4104https://repository.hanyang.ac.kr/handle/20.500.11754/112880
- ISSN
- 1548-7091; 1548-7105
- DOI
- 10.1038/NMETH.4104
- Appears in Collections:
- COLLEGE OF MEDICINE[S](의과대학) > MEDICINE(의학과) > Articles
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