Journal
SCIENTIFIC REPORTS
Volume 9, Issue -, Pages -Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/s41598-019-41356-1
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Funding
- National Research Foundation of Korea (NRF) [2018M3A9H3022412]
- Next Generation BioGreen 21 Program [PJ01319301]
- Technology Innovation Program - Ministry of Trade, Industry and Energy [20000158]
- Korea Healthcare technology RD Project [HI16C1012]
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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.
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