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CRISPR-Cas Genome Editing in the Cellulolytic Bacterium Clostridium thermocellum (C. thermocellum)

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Clostridium thermocellum is an anaerobic thermophile that can efficiently degrade lignocellulosic biomass and directly convert it into value-added products such as ethanol. The cellulolytic and ethanologenic capabilities of C. thermocellum make it an excellent candidate for consolidated bioprocessing (CBP) for industrial production, where biomass degradation and fermentation occur simultaneously. In this organism, strain development for effective CBP has traditionally been hindered by the lack of genetic tools. Here, we detail our efficient two-step CRISPR-Cas genome editing protocol for C. thermocellum, using both the native Type I-B CRISPR-Cas system and an exogenous Type-II CRISPR system from Geobacillus stearothermophilus. As recombination is limiting in C. thermocellum genome engineering, we highlight effective thermophilic recombinases necessary to improve genome editing in these systems. We additionally provide design rules for the repair template and synthetic guide RNA (gRNA) for each system. Using these newly developed CRISPR and recombineering tools, targeted C. thermocellum engineering will substantiate efforts toward CBP strain development in industrial applications.

Research Organization:
National Renewable Energy Laboratory (NREL), Golden, CO (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
DOE Contract Number:
AC36-08GO28308
OSTI ID:
1986067
Report Number(s):
NREL/CH-2700-78415; MainId:32332; UUID:f3e674f1-13d3-4a45-b65f-28996ad2e90a; MainAdminID:18858
Country of Publication:
United States
Language:
English

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