|
Type |
Poster Presentation |
Area |
Life Chemistry |
Room No. |
Grand Ballroom |
Time |
10월 19일 (금요일) 11:00~12:30 |
Code |
LIFE.P-430 |
Subject |
Construction of non-canonical PAM-targeting adenosine base editors by restriction enzyme-free DNA cloning using CRISPR-Cas9 |
Authors |
Youkyeong Jeong, Jihyeon Yu1, Sangsu Bae2,* Chemistry, Hanyang University, Korea 1Department of chemistry, Hanyang University, Korea 2Department of Chemistry, Hanyang University, Korea |
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 develop 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 and VRER), which target non-canonical NGA and NGCG protospacer-adjacent motif (PAM) sequences, fused with adenosine deaminases for adenine base editing (named VQR-ABE and VRER-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 and NGCG PAMs, expanding the targetable range of the adenine base editing. |
E-mail |
dpflapfl@naver.com |
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