A repackaged CRISPR platform increases homology-directed repair for yeast engineering.
Nat Chem Biol
; 18(1): 38-46, 2022 01.
Article
em En
| MEDLINE
| ID: mdl-34711982
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Saccharomyces cerevisiae
/
Engenharia Genética
/
Sistemas CRISPR-Cas
Tipo de estudo:
Prognostic_studies
Idioma:
En
Revista:
Nat Chem Biol
Assunto da revista:
BIOLOGIA
/
QUIMICA
Ano de publicação:
2022
Tipo de documento:
Article
País de afiliação:
Estados Unidos