CRISPR-Cas9-mediated pinpoint microbial genome editing aided by target-mismatched sgRNAs
DC Field | Value | Language |
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dc.contributor.author | Lee, Ho Joung | - |
dc.contributor.author | Kim, Hyun Ju | - |
dc.contributor.author | Lee, Sang Jun | - |
dc.date.available | 2020-07-13T04:21:40Z | - |
dc.date.issued | 2020-05 | - |
dc.identifier.issn | 1088-9051 | - |
dc.identifier.issn | 1549-5469 | - |
dc.identifier.uri | https://scholarworks.bwise.kr/cau/handle/2019.sw.cau/41782 | - |
dc.description.abstract | Genome editing has been revolutionized by the CRISPR-Cas9 system. CRISPR-Cas9 is composed of single-molecular guide RNA (sgRNA) and a proteinaceous Cas9 nuclease, which recognizes a specific target sequence and a protospacer adjacent motif (PAM) sequence and, subsequently, cleaves the targeted DNA sequence. This CRISPR-Cas9 system has been used as an efficient negative-selection tool to cleave unedited or unchanged target DNAs during site-specific mutagenesis and, consequently, obtain microbial cells with desired mutations. This study aimed to investigate the genome editing efficiency of the CRISPR-Cas9 system for in vivo oligonucleotide-directed mutagenesis in bacteria. This system successfully introduced two-to four-base mutations in galK in Escherichia coli with high editing efficiencies (81%-86%). However, single-point mutations (T504A or C578A) were rarely introduced with very low editing efficiencies (<3%), probably owing to mismatch tolerance. To resolve this issue, we designed one- or two-base mismatches in the sgRNA sequence to recognize target sequences in galK in E. coli. A single-point nucleotide mutation (T504A or C578A in the galKgene) was successfully introduced in 36%-95% of negatively selected E. coli cells using single-base mismatched sgRNAs. Sixteen targets were randomly selected through genome-wide single-base editing experiments using mismatched sgRNAs. Consequently, out of 48 desired single-base mutations, 25 single bases were successfully edited, using mismatched sgRNAs. Finally, applicable design rules for target-mismatched sgRNAs were provided for single-nucleotide editing in microbial genomes. | - |
dc.format.extent | 8 | - |
dc.language | 영어 | - |
dc.language.iso | ENG | - |
dc.publisher | COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT | - |
dc.title | CRISPR-Cas9-mediated pinpoint microbial genome editing aided by target-mismatched sgRNAs | - |
dc.type | Article | - |
dc.identifier.doi | 10.1101/gr.257493.119 | - |
dc.identifier.bibliographicCitation | GENOME RESEARCH, v.30, no.5, pp 768 - 775 | - |
dc.description.isOpenAccess | Y | - |
dc.identifier.wosid | 000536952700010 | - |
dc.identifier.scopusid | 2-s2.0-85085586137 | - |
dc.citation.endPage | 775 | - |
dc.citation.number | 5 | - |
dc.citation.startPage | 768 | - |
dc.citation.title | GENOME RESEARCH | - |
dc.citation.volume | 30 | - |
dc.type.docType | Article | - |
dc.publisher.location | 미국 | - |
dc.subject.keywordPlus | CRISPR-CAS SYSTEMS | - |
dc.subject.keywordPlus | CLASSIFICATION | - |
dc.subject.keywordPlus | REPEATS | - |
dc.subject.keywordPlus | GENE | - |
dc.subject.keywordPlus | DNA | - |
dc.relation.journalResearchArea | Biochemistry & Molecular Biology | - |
dc.relation.journalResearchArea | Biotechnology & Applied Microbiology | - |
dc.relation.journalResearchArea | Genetics & Heredity | - |
dc.relation.journalWebOfScienceCategory | Biochemistry & Molecular Biology | - |
dc.relation.journalWebOfScienceCategory | Biotechnology & Applied Microbiology | - |
dc.relation.journalWebOfScienceCategory | Genetics & Heredity | - |
dc.description.journalRegisteredClass | scie | - |
dc.description.journalRegisteredClass | scopus | - |
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