Nanopore sequencing improves construction of customized CRISPR-based gene activation libraries
Handing Wang Heng Yih Tan Jiazhang Lian Kang Zhou
Handing Wang
Heng Yih Tan
Jiazhang Lian
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Alternative Title
Abstract
Clustered 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.
Keywords
CRISPR interference, Nanopore sequencing
Source Title
Biotechnology and Bioengineering
Publisher
John Wiley & Sons, Inc.
Series/Report No.
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Rights
Attribution-NonCommercial-NoDerivatives 4.0 International
Date
2024-01-31
DOI
https://doi.org/10.1002/bit.28664
Type
Article
Additional Links
Related Datasets
https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA1049995