Exploiting endogenous CRISPR-Cas system for multiplex genome editing in Clostridium tyrobutyricum and engineer the strain for high-level butanol production

Abstract

Although CRISPR-Cas9/Cpf1 have been employed as powerful genome engineering tools, heterologous CRISPR-Cas9/Cpf1 are often difficult to introduce into bacteria and archaea due to their severe toxicity. Since most prokaryotes harbor native CRISPR-Cas systems, genome engineering can be achieved by harnessing these endogenous immune systems. Here, we report the exploitation of Type I-B CRISPR-Cas of Clostridium tyrobutyricum for genome engineering. In silico CRISPR array analysis and plasmid interference assay revealed that TCA or TCG at the 5'-end of the protospacer was the functional protospacer adjacent motif (PAM) for CRISPR targeting. With a lactose inducible promoter for CRISPR array expression, we significantly decreased the toxicity of CRISPR-Cas and enhanced the transformation efficiency, and successfully deleted spo0A with an editing efficiency of 100%. We further evaluated effects of the spacer length on genome editing efficiency. Interestingly, spacers ≤ 20 nt led to unsuccessful transformation consistently, likely due to severe off-target effects; while a spacer of 30-38 nt is most appropriate to ensure successful transformation and high genome editing efficiency. Moreover, multiplex genome editing for the deletion of spo0A and pyrF was achieved in a single transformation, with an editing efficiency of up to 100%. Finally, with the integration of the alcohol dehydrogenase gene (adhE1 or adhE2) to replace cat1 (the key gene responsible for butyrate production and previously could not be deleted), two mutants were created for n-butanol production, with the butanol titer reached historically record high of 26.2 g/L in a batch fermentation. Altogether, our results demonstrated the easy programmability and high efficiency of endogenous CRISPR-Cas. The developed protocol herein has a broader applicability to other prokaryotes containing endogenous CRISPR-Cas systems. C. tyrobutyricum could be employed as an excellent platform to be engineered for biofuel and biochemical production using the CRISPR-Cas based genome engineering toolkit.

Characterization and repurposing of the endogenous Type I-F CRISPR-Cas system ofZymomonas mobilisfor genome engineering

“…To date, genome engineering with the already developed native Type I CRISPR-based toolkits can be achieved at generally high efficiencies (31,32,35). However, based on the specific genetic backgrounds of various microorganisms, several attempts were made on the optimization of the native CRISPR-based genome engineering toolkits, aiming at high engineering efficiencies.…”

“…We adopted the same strategy in this study. In another study where the endogenous Type I-B of Clostridium tyrobutyricum was exploited for genome engineering, Zhang and co-authors tested the spacer length of the artificial CRISPR on the efficiencies of plasmid transformation and genome engineering (35).…”

“…Although we have succeeded in various genome engineering purposes with the Type I-F CRISPR-Cas system in Z. mobilis, we also noticed that the engineering efficiencies was much lower (6.79-37.5%) than that obtained by repurposing other native Type I systems (31,32,35). This left sufficient room for its further improvement and we therefore sought to update this toolkit to be a more efficient one.…”

“…In recent years, several endogenous CRISPR-Cas systems, with the emphasis on Type I, have been harnessed as an alternative strategy to replace the Class 2 CRISPR-based toolkits for prokaryotic engineering in several archaea and bacteria (31)(32)(33)(34)(35)(36). Notably, an endogenous Type I-B system was developed as an efficient genome editing toolkit for Clostridium tyrobutyricum, an important industrial microorganism for acetone-butanol-ethanol (ABE) production.…”

“…Notably, an endogenous Type I-B system was developed as an efficient genome editing toolkit for Clostridium tyrobutyricum, an important industrial microorganism for acetone-butanol-ethanol (ABE) production. Using the toolkit, C. tyrobutyricum was engineered for n-butanol production and the engineered strains produced butanol with the titer of as historically record high as 26.2 g/L in a batch fermentation (35). However, to the best of our knowledge, this represents thus far the only one native CRISPR-based toolkit for an industrial non-model microorganism.…”