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dc.contributor.author유지현-
dc.date.accessioned2019-08-22T02:37:06Z-
dc.date.available2019-08-22T02:37:06Z-
dc.date.issued2019-03-
dc.identifier.citationSCIENTIFIC REPORTS, v. 9, no. 4939en_US
dc.identifier.issn2045-2322-
dc.identifier.urihttps://www.nature.com/articles/s41598-019-41356-1-
dc.identifier.urihttps://repository.hanyang.ac.kr/handle/20.500.11754/108895-
dc.description.abstractMolecular 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.en_US
dc.description.sponsorshipThis work was supported by National Research Foundation of Korea (NRF) Grants (no. 2018M3A9H3022412), Next Generation BioGreen 21 Program grant no. PJ01319301, Technology Innovation Program funded by the Ministry of Trade, Industry and Energy (no. 20000158), and Korea Healthcare technology R&D Project grant no. HI16C1012 to S.B.en_US
dc.language.isoenen_US
dc.publisherNATURE PUBLISHING GROUPen_US
dc.subjectCAS9en_US
dc.subjectRNAen_US
dc.subjectGUIDEen_US
dc.titleConstruction of non-canonical PAM-targeting adenosine base editors by restriction enzyme-free DNA cloning using CRISPR-Cas9en_US
dc.typeArticleen_US
dc.relation.volume9-
dc.identifier.doi10.1038/s41598-019-41356-1-
dc.relation.page4939-4939-
dc.relation.journalSCIENTIFIC REPORTS-
dc.contributor.googleauthorJeong, You Kyeong-
dc.contributor.googleauthorYu, Jihyeon-
dc.contributor.googleauthorBae, Sangsu-
dc.relation.code2019002548-
dc.sector.campusS-
dc.sector.daehakRESEARCH INSTITUTE[S]-
dc.sector.departmentRESEARCH INSTITUTE FOR CONVERGENCE OF BASIC SCIENCES-
dc.identifier.pidmuner00-


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