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CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art
Published on: May 28, 2019
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A repackaged CRISPR platform increases homology-directed repair for yeast engineering
Deon Ploessl1,2, Yuxin Zhao1,2, Mingfeng Cao1,2
1Department of Chemical and Biological Engineering, Iowa State University, Ames, IA, USA.
Nature Chemical Biology
|October 29, 2021
Summary
This study introduces the LINEAR CRISPR system to enhance homology-directed repair (HDR) in yeasts, enabling precise genome editing without disrupting nonhomologous end joining (NHEJ). This advances microbial factory engineering for compound production.
Area of Science:
- Synthetic Biology
- Molecular Biology
- Genetics
Background:
- Inefficient homology-directed repair (HDR) limits CRISPR-Cas9 genome editing in organisms favoring nonhomologous end joining (NHEJ) for DNA double-strand breaks (DSBs).
- Existing methods to improve HDR often involve disrupting NHEJ, which can have unintended consequences.
Purpose of the Study:
- To develop a novel CRISPR platform that enhances HDR without disrupting NHEJ.
- To enable precise genome editing in NHEJ-proficient organisms.
- To leverage both HDR and NHEJ pathways for microbial engineering.
Main Methods:
- Developed the CRISPR platform-lowered indel nuclease system enabling accurate repair (LINEAR).
- Tested LINEAR in four NHEJ-proficient yeast strains.
- Utilized LINEAR for precision editing and NHEJ for random integration to engineer a microbial factory.
Main Results:
- Achieved significantly enhanced HDR rates (67-100%) using LINEAR compared to conventional CRISPR platforms in yeasts.
- Demonstrated the ability to identify genomic loci with favorable expression dynamics for heterologous pathways while preserving NHEJ.
- Successfully engineered and optimized a microbial factory for (S)-norcoclaurine production using LINEAR and NHEJ.
Conclusions:
- The LINEAR system offers a precise genome editing solution for NHEJ-proficient organisms.
- Leveraging antagonistic DNA repair pathways (HDR and NHEJ) expands capabilities in microbial factory development.
- This approach broadens the toolkit for engineering microbes for industrial applications.
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