Single and multiplexed gene repression in solventogenic Clostridium via Cas12a-based CRISPR interference.
Synth Syst Biotechnol
; 8(1): 148-156, 2023 Mar.
Article
em En
| MEDLINE
| ID: mdl-36687471
The Gram-positive, spore-forming, obligate anaerobic firmicute species that make up the Clostridium genus have broad feedstock consumption capabilities and produce value-added metabolic products, but genetic manipulation is difficult, limiting their broad appeal. CRISPR-Cas systems have recently been applied to Clostridium species, primarily using Cas9 as a counterselection marker in conjunction with plasmid-based homologous recombination. CRISPR interference is a method that reduces gene expression of specific genes via precision targeting of a nuclease deficient Cas effector protein. Here, we develop a dCas12a-based CRISPR interference system for transcriptional gene repression in multiple mesophilic Clostridium species. We show the Francisella novicida Cas12a-based system has a broader applicability due to the low GC content in Clostridium species compared to CRISPR Cas systems derived from other bacteria. We demonstrate >99% reduction in transcript levels of targeted genes in Clostridium acetobutylicum and >75% reduction in Clostridium pasteurianum. We also demonstrate multiplexed repression via use of a single synthetic CRISPR array, achieving 99% reduction in targeted gene expression and elucidating a unique metabolic profile for their reduced expression. Overall, this work builds a foundation for high throughput genetic screens without genetic editing, a key limitation in current screening methods used in the Clostridium community.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Idioma:
En
Revista:
Synth Syst Biotechnol
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
Estados Unidos