4 0

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/s41598-019-41356-1http://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
Export
RIS (EndNote)
XLS (Excel)
XML


qrcode

Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

BROWSE