Development of a CRISPR-Cas12a system for efficient genome engineering in clostridia

Abstract

Clostridium species have gained attention in industrial and medical applications, and the development of genetic tools has enabled the advancement of the CRISPR-Cas systems for these bacteria. Compared to the primarily used Cas9 from Streptococcus pyogenes , the utilization of Cas12a (previously known as Cpf1) proteins remains incomplete in clostridia, although they exhibit potential advantages, including T-rich recognition for Clostridium genomes and lower toxicity. In this study, we expanded the CRISPR-Cas tools in clostridia by establishing a CRISPR-Cas12a system with two different cas12a genes ( Ascas12a from Acidaminococcus and Fncas12a from Francisella novicida ). The optimized tetracycline-inducible systems were initially determined by the glucuronidase reporter and were used to drive expression of the cas12a genes and crRNAs. Our results demonstrate that the CRISPR-FnCas12a system offers flexible target selection in clostridia, and a specific folding pattern of the precursor crRNA is important to enable high mutation generation efficiency. By using sacB (encoding levansucrase) as a negative marker for plasmid curing and determining the optimal size of the donor DNA template for gene integration in the CRISPR-FnCas12a system, we achieved highly efficient and rapid genome modification, exemplified by the successful engineering of clostridia ( Clostridium butyricum and Clostridium sporogenes ) to produce near-infrared fluorescence from biliverdin and hemin. IMPORTANCE Continued efforts in developing the CRISPR-Cas systems will further enhance our understanding and utilization of Clostridium species. This study demonstrates the development and application of a genome-engineering tool in two Clostridium strains, Clostridium butyricum and Clostridium sporogenes , which have promising potential as probiotics and oncolytic agents. Particular attention was given to the folding of precursor crRNA and the role of this process in off-target DNA cleavage by Cas12a. The results provide the guidelines necessary for efficient genome engineering using this system in clostridia. Our findings not only expand our fundamental understanding of genome-engineering tools in clostridia but also improve this technology to allow use of its full potential in a plethora of biotechnological applications.

Multiplex genetic manipulations in Clostridium butyricum and Clostridium sporogenes to secrete recombinant antigen proteins for oral-spore vaccination

“…In order to create plasmid-free clostridia for regulatory approval of COSV, the PyrE loci in the genomes of C. butyricum and C. sporogenes were replaced with high-expression cassettes carrying NY-ESO-1 and CDP-NY-ESO-1, utilizing the previously developed CRISPR-Cas12a system [ 31 ]. The introduction of uracil auxotrophy in the pyrE -deficient clostridia is known to ensure CBT bio-containment [ 16 ].…”

“…For the construction of genome integration plasmids targeting PyrE loci in clostridia, target fragments were amplified and ligated into the pP fet Fn-Target_v2 entry vector of the CRISPR-FnCas12a system [ 31 ] using Golden Gate assembly (BsmBI-v2, NEB). After sequencing confirmation, the ligated vectors were digested by AatII and SalI enzymes (NEB), resulting in a 6.5-kb backbone.…”

“…All plasmids were introduced into E. coli through heat shock, followed by conjugation ( E. coli S17-1) into clostridia as previously described [ 31 ].…”

“…CRISPR-FnCas12a-mediated genome engineering in clostridia was performed as we previously described [ 31 ]. Briefly, the entry vector pP IPL12 -FnCas12a was conjugated into clostridia, followed by the conjugation of the corresponding genome integration plasmids into these clostridia.…”

Clostridium Bacteria: Harnessing Tumour Necrosis for Targeted Gene Delivery

“…Alternative endonucleases (e.g. Cas12a, also known as Cpf1) have since been developed to overcome limitations of Cas9 and may prove more advantageous for genomic modification of Clostridium in future [ 35 – 37 ].…”