A repackaged CRISPR platform increases homology-directed repair for yeast engineering.
Journal
Nature chemical biology
ISSN: 1552-4469
Titre abrégé: Nat Chem Biol
Pays: United States
ID NLM: 101231976
Informations de publication
Date de publication:
01 2022
01 2022
Historique:
received:
10
02
2021
accepted:
09
09
2021
pubmed:
30
10
2021
medline:
23
2
2022
entrez:
29
10
2021
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 34711982
doi: 10.1038/s41589-021-00893-5
pii: 10.1038/s41589-021-00893-5
doi:
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
38-46Informations de copyright
© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.
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