CRISPR‐based genome editing and expression control systems in Clostridium acetobutylicum and Clostridium beijerinckii

Abstract

Solventogenic clostridia are important industrial microorganisms that produce various chemicals and fuels. Effective genetic tools would facilitate physiological studies aimed both at improving our understanding of metabolism and optimizing solvent productivity through metabolic engineering. Here we have developed an all-in-one, CRISPR-based genome editing plasmid, pNICKclos, that can be used to achieve successive rounds of gene editing in Clostridium acetobutylicum ATCC 824 and Clostridium beijerinckii NCIMB 8052 with efficiencies varying from 6.7% to 100% and 18.8% to 100%, respectively. The plasmid specifies the requisite target-specific guide RNA, the gene encoding the Streptococcus pyogenes Cas9 nickase and the genome editing template encompassing the gene-specific homology arms. It can be used to create single target mutants within three days, with a further two days required for the curing of the pNICKclos plasmid ready for a second round of mutagenesis. A S. pyogenes dCas9-mediated gene regulation control system, pdCASclos, was also developed and used in a CRISPRi strategy to successfully repress the expression of spo0A in C. acetobutylicum and C. beijerinckii. The combined application of the established high efficiency CRISPR-Cas9 based genome editing and regulation control systems will greatly accelerate future progress in the understanding and manipulation of metabolism in solventogenic clostridia.

CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum

“…In contrast to a double-strand break catalysed by native Cas9, the single-strand nick created by Cas9 nickase is prone to repair and thus increases the number of edited cells22. To create an Fn Cpf1 nickase, we prepared an R1218A mutation that corresponded to the R1226A mutant of Acidaminococcus sp.…”

Development of a High-Efficiency Transformation Method and Implementation of Rational Metabolic Engineering for the Industrial Butanol Hyperproducer Clostridium saccharoperbutylacetonicum Strain N1-4

“…Recent advances in the genetic manipulation of clostridia (primarily C. acetobutylicum ATCC 824 and C. beijerinckii NCIMB 8052) have afforded several methods for targeted gene disruption, including (i) heterologous expression of mobile group II introns with the ClosTron system (36), (ii) allelic exchange via homologous recombination (37,38), and very recently, (iii) clustered regularly interspaced short palindromic repeat(s) (CRISPR)-Cas9 genome editing (39)(40)(41). Given the observed instability and off-target effects associated with mobile group II introns (42) as well as the start-up time required to adapt an efficient CRISPR-Cas9 system for use in C. saccharoperbutylacetonicum N1-4, we chose here to pursue a double-crossover-based allelic exchange (DCAE) method.…”

“…This study serves as a foundation for future metabolic engineering efforts of this prominent solvent producer to not only improve fermentation performance metrics (e.g., solvent titers, productivities, and yields, oxygen tolerance, substrate specificity, cell viability), but also to serve as a platform for the production of other high-value products (e.g., higher alcohols and petrochemical precursors). Additionally, this study opens the door for advanced genetic manipulations of this organism using recent achievements in adapting CRISPR/Cas9 systems for use in clostridia (40,41). Before this is possible, future studies are required to identify additional promoters, antibiotic markers, and counterselection methods able to function in C. saccharoperbutylacetonicum N1-4.…”

Consolidated bioprocessing for butanol production of cellulolytic Clostridia: development and optimization

“…In the post‐genome era, synthetic biology techniques provide resources and approaches for CBP construction and optimization. However, inefficient DNA repair and low plasmid transformation efficiency lead to lack of synthetic biology tools for Clostridium (Li et al ., ,b), which severely delays CBP development. To accelerate the bottleneck breakthrough, roadmaps or technical guides for the genetic advancement in Clostridium have been extensively suggested and reviewed (Pyne et al ., ; Minton et al ., ; Joseph et al ., ).…”

“…(,b,c) and Li et al . (,b) introduced Cas9 nickase to C. cellulolyticum and solventogenic Clostridia, respectively, which resulted in a single nick, which triggered homologous recombination and enhanced genome editing efficiency.…”