122nd General Meeting of the KCS

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