Construction of non-canonical PAM-targeting adenosine base editors by restriction enzyme-free DNA cloning using CRISPR-Cas9
- Title
- Construction of non-canonical PAM-targeting adenosine base editors by restriction enzyme-free DNA cloning using CRISPR-Cas9
- Author
- 유지현
- Keywords
- CAS9; RNA; GUIDE
- Issue Date
- 2019-03
- Publisher
- NATURE PUBLISHING GROUP
- Citation
- SCIENTIFIC REPORTS, v. 9, no. 4939
- Abstract
- Molecular cloning is an essential technique in molecular biology and biochemistry, but it is frequently laborious when adequate restriction enzyme recognition sites are absent. Cas9 endonucleases can induce site-specific DNA double-strand breaks at sites homologous to their guide RNAs, rendering an alternative to restriction enzymes. Here, by combining DNA cleavage via a Cas9 endonuclease and DNA ligation via Gibson assembly, we demonstrate a precise and practical DNA cloning method for replacing part of a backbone plasmid. We first replaced a resistance marker gene as a proof of concept and next generated DNA plasmids that encode engineered Cas9 variants (VQR, VRER and SpCas9-NG), which target non-canonical NGA, NGCG and NG protospacer-adjacent motif (PAM) sequences, fused with adenosine deaminases for adenine base editing (named VQR-ABE, VRER-ABE and NG-ABE, respectively). Ultimately, we confirmed that the re-constructed plasmids can successfully convert adenosine to guanine at endogenous target sites containing the non-canonical NGA, NGCG and NG PAMs, expanding the targetable range of the adenine base editing.
- URI
- https://www.nature.com/articles/s41598-019-41356-1https://repository.hanyang.ac.kr/handle/20.500.11754/108895
- ISSN
- 2045-2322
- DOI
- 10.1038/s41598-019-41356-1
- Appears in Collections:
- RESEARCH INSTITUTE[S](부설연구소) > ETC
- Files in This Item:
- Construction of non-canonical PAM-targeting adenosine base editors by restriction enzyme-free DNA cloning using CRISPR-Cas9.pdfDownload
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