Please use this identifier to cite or link to this item: https://doi.org/https://doi.org/10.1002/bit.28664
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dc.titleNanopore sequencing improves construction of customized CRISPR-based gene activation libraries
dc.contributor.authorHanding Wang
dc.contributor.authorHeng Yih Tan
dc.contributor.authorJiazhang Lian
dc.contributor.authorKang Zhou
dc.contributor.editorHal, Alper
dc.date.accessioned2024-05-02T02:33:29Z
dc.date.available2024-05-02T02:33:29Z
dc.date.issued2024-01-31
dc.identifier.citationHanding Wang, Heng Yih Tan, Jiazhang Lian, Kang Zhou (2024-01-31). Nanopore sequencing improves construction of customized CRISPR-based gene activation libraries. Biotechnology and Bioengineering 121 (5) : 1543-1553. ScholarBank@NUS Repository. https://doi.org/https://doi.org/10.1002/bit.28664
dc.identifier.urihttps://scholarbank.nus.edu.sg/handle/10635/248190
dc.description.abstractClustered regularly interspaced short palindromic repeats (CRISPR)‐based screening has emerged as a powerful tool for identifying new gene targets for desired cellular phenotypes. The construction of guide RNA (gRNA) pools largely determines library quality and is usually performed using Golden Gate assembly or Gibson assembly. To date, library construction methods have not been systematically compared, and the quality check of each batch has been slow. In this study, an in‐house nanopore sequencing workflow was established for assessing the current methods of gRNA pool construction. The bias of pool construction was reduced by employing the polymerase‐mediated non‐amplifying method. Then, a small gRNA pool was utilized to characterize stronger activation domains, specifically MED2 (a subunit of mediator complex) and HAP4 (a heme activator protein), as well as to identify better gRNA choices for dCas12a‐based gene activation in Saccharomyces cerevisiae. Furthermore, based on the better CRISPRa tool identified in this study, a custom gRNA pool, which consisted of 99 gRNAs targeting central metabolic pathways, was designed and employed to screen for gene targets that could improve ethanol utilization in S. cerevisiae. The nanopore sequencing‐based workflow demonstrated here should provide a cost‐effective approach for assessing the quality of customized gRNA library, leading to faster and more efficient genetic and metabolic engineering in S. cerevisiae.
dc.language.isoen
dc.publisherJohn Wiley & Sons, Inc.
dc.rightsAttribution-NonCommercial-NoDerivatives 4.0 International
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0/
dc.subjectCRISPR interference
dc.subjectNanopore sequencing
dc.typeArticle
dc.contributor.departmentCHEMICAL & BIOMOLECULAR ENGINEERING
dc.description.doihttps://doi.org/10.1002/bit.28664
dc.description.sourcetitleBiotechnology and Bioengineering
dc.description.volume121
dc.description.issue5
dc.description.page1543-1553
dc.published.statePublished
dc.grant.fundingagencyNational Research Foundation of Singapore
dc.grant.fundingagencyNatural Science Foundation of Zhejiang Province
dc.grant.fundingagencyNatural Science Foundation of China
dc.relation.datasethttps://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA1049995
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