A repackaged CRISPR platform increases homology-directed repair for yeast engineering
Date
2021-10-28
Authors
Ploessl, Deon
Zhao, Yuxin
Cao, Mingfeng
Ghosh, Saptarshi
Lopez, Carmen
Sayadi, Maryam
Chudalayandi, Sivanandan
Huang, Lei
Gustafson, Marissa
Major Professor
Advisor
Committee Member
Journal Title
Journal ISSN
Volume Title
Publisher
Springer Nature
Abstract
Inefficient homology-directed repair (HDR) constrains CRISPR–Cas9 genome editing in organisms that preferentially employ nonhomologous end joining (NHEJ) to fix DNA double-strand breaks (DSBs). Current strategies used to alleviate NHEJ proficiency involve NHEJ disruption. To confer precision editing without NHEJ disruption, we identified the shortcomings of the conventional CRISPR platforms and developed a CRISPR platform—lowered indel nuclease system enabling accurate repair (LINEAR)—which enhanced HDR rates (to 67–100%) compared to those in previous reports using conventional platforms in four NHEJ-proficient yeasts. With NHEJ preserved, we demonstrate its ability to survey genomic landscapes, identifying loci whose spatiotemporal genomic architectures yield favorable expression dynamics for heterologous pathways. We present a case study that deploys LINEAR precision editing and NHEJ-mediated random integration to rapidly engineer and optimize a microbial factory to produce (S)-norcoclaurine. Taken together, this work demonstrates how to leverage an antagonizing pair of DNA DSB repair pathways to expand the current collection of microbial factories.
Series Number
Journal Issue
Is Version Of
Versions
Series
Type
article
Comments
This is a post-peer-review, pre-copyedit version of an article published in Nature Chemical Biology. The final authenticated version is available online at DOI: 10.1038/s41589-021-00893-5. Copyright © 2021, The Author(s). Posted with permission.