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A Universal Protocol for Large-scale gRNA Library Production from any DNA Source
Published on: December 6, 2017
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Nanopore sequencing improves construction of customized CRISPR-based gene activation libraries
Handing Wang1,2, Heng Yih Tan2, Jiazhang Lian1,3
1Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, China.
Biotechnology and Bioengineering
|January 31, 2024
Summary
This study introduces a fast nanopore sequencing method to assess CRISPR guide RNA (gRNA) pools for yeast gene editing. This improves the efficiency of genetic and metabolic engineering in Saccharomyces cerevisiae.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- CRISPR screening is vital for identifying gene targets for cellular phenotypes.
- Guide RNA (gRNA) pool construction methods like Golden Gate and Gibson assembly lack systematic comparison and efficient quality control.
- Current quality checks for gRNA libraries are time-consuming.
Purpose of the Study:
- To establish and validate an in-house nanopore sequencing workflow for assessing gRNA pool construction methods.
- To reduce bias in gRNA pool construction using a polymerase-mediated non-amplifying method.
- To identify optimal gene activation tools and gRNAs for CRISPR activation in Saccharomyces cerevisiae and screen for improved ethanol utilization.
Main Methods:
- Development of an in-house nanopore sequencing workflow for gRNA pool quality assessment.
- Utilized a polymerase-mediated non-amplifying method to minimize pool construction bias.
- Employed CRISPRa screening with dCas12a in Saccharomyces cerevisiae to characterize activation domains and optimize gRNAs.
- Designed and screened a custom gRNA pool targeting central metabolic pathways for enhanced ethanol utilization.
Main Results:
- The nanopore sequencing workflow provides a cost-effective and rapid method for assessing gRNA library quality.
- Identified stronger activation domains (MED2, HAP4) and superior gRNAs for dCas12a-based gene activation.
- Successfully screened a custom gRNA library to identify gene targets for improved ethanol utilization in S. cerevisiae.
- Demonstrated reduced bias in gRNA pool construction using the non-amplifying method.
Conclusions:
- The developed nanopore sequencing workflow significantly enhances the speed and efficiency of gRNA library quality control.
- This approach facilitates faster and more effective genetic and metabolic engineering in Saccharomyces cerevisiae.
- The study provides a valuable tool for optimizing CRISPR-based screening and engineering applications.
