New Target Gene Screening Using Shortened and Random sgRNA Libraries in Microbial CRISPR Interference
DC Field | Value | Language |
---|---|---|
dc.contributor.author | Jeong, Song Hee | - |
dc.contributor.author | Kim, Hyun Ju | - |
dc.contributor.author | Lee, Sang Jun | - |
dc.date.accessioned | 2023-04-17T07:42:44Z | - |
dc.date.available | 2023-04-17T07:42:44Z | - |
dc.date.issued | 2023-03 | - |
dc.identifier.issn | 2161-5063 | - |
dc.identifier.uri | https://scholarworks.bwise.kr/cau/handle/2019.sw.cau/66397 | - |
dc.description.abstract | CRISPR interference (CRISPRi) screening has been used for identification of target genes related to specific phenotypes using single-molecular guide RNA (sgRNA) libraries. In CRISPRi screening, the sizes of random sgRNA libraries contained with the original target recognition sequences are large (∼1012). Here, we demonstrated that the length of the target recognition sequence (TRS) can be shortened in sgRNAs from the original 20 nucleotides (N20) to 9 nucleotides (N9) that is still sufficient for dCas9 to repress target genes in the xylose operon of Escherichia coli, regardless of binding to a promoter or open reading frame region. Based on the results, we constructed random sgRNA plasmid libraries with 5′-shortened TRS lengths, and identified xylose metabolic target genes by Sanger sequencing of sgRNA plasmids purified from Xyl- phenotypic cells. Next, the random sgRNA libraries were harnessed to screen for target genes to enhance violacein pigment production in synthetic E. coli cells. Seventeen target genes were selected by analyzing the redundancy of the TRS in sgRNA plasmids in dark purple colonies. Among them, seven genes (tyrR, pykF, cra, ptsG, pykA, sdaA, and tnaA) have been known to increase the intracellular l-tryptophan pool, the precursor of a violacein. Seventeen cells with a single deletion of each target gene exhibited a significant increase in violacein production. These results indicate that using shortened random TRS libraries for CRISPRi can be simple and cost-effective for phenotype-based target gene screening. © 2023 The Authors. Published by American Chemical Society. | - |
dc.format.extent | 9 | - |
dc.language | 영어 | - |
dc.language.iso | ENG | - |
dc.publisher | American Chemical Society | - |
dc.title | New Target Gene Screening Using Shortened and Random sgRNA Libraries in Microbial CRISPR Interference | - |
dc.type | Article | - |
dc.identifier.doi | 10.1021/acssynbio.2c00595 | - |
dc.identifier.bibliographicCitation | ACS Synthetic Biology, v.12, no.3, pp 800 - 808 | - |
dc.description.isOpenAccess | Y | - |
dc.identifier.wosid | 000933962200001 | - |
dc.identifier.scopusid | 2-s2.0-85148349440 | - |
dc.citation.endPage | 808 | - |
dc.citation.number | 3 | - |
dc.citation.startPage | 800 | - |
dc.citation.title | ACS Synthetic Biology | - |
dc.citation.volume | 12 | - |
dc.type.docType | Article | - |
dc.publisher.location | 미국 | - |
dc.subject.keywordAuthor | CRISPR interference | - |
dc.subject.keywordAuthor | deactivated Cas9 | - |
dc.subject.keywordAuthor | phenotype-based target screening | - |
dc.subject.keywordAuthor | random library | - |
dc.subject.keywordAuthor | shortened sgRNA | - |
dc.subject.keywordAuthor | target recognition sequence | - |
dc.subject.keywordAuthor | violacein | - |
dc.subject.keywordPlus | SEQUENCE-SPECIFIC CONTROL | - |
dc.subject.keywordPlus | ESCHERICHIA-COLI | - |
dc.subject.keywordPlus | VIOLACEIN | - |
dc.subject.keywordPlus | STRAIN | - |
dc.relation.journalResearchArea | Biochemistry & Molecular Biology | - |
dc.relation.journalWebOfScienceCategory | Biochemical Research Methods | - |
dc.description.journalRegisteredClass | scie | - |
dc.description.journalRegisteredClass | scopus | - |
Items in ScholarWorks are protected by copyright, with all rights reserved, unless otherwise indicated.
84, Heukseok-ro, Dongjak-gu, Seoul, Republic of Korea (06974)02-820-6194
COPYRIGHT 2019 Chung-Ang University All Rights Reserved.
Certain data included herein are derived from the © Web of Science of Clarivate Analytics. All rights reserved.
You may not copy or re-distribute this material in whole or in part without the prior written consent of Clarivate Analytics.