Extending CRISPR-Cas9 Technology from Genome Editing to Transcriptional Engineering in the Genus Clostridium

Abstract

The discovery and exploitation of the prokaryotic adaptive immunity system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins have revolutionized genetic engineering. CRISPR-Cas tools have enabled extensive genome editing as well as efficient modulation of the transcriptional program in a multitude of organisms. Progress in the development of genetic engineering tools for the genus Clostridium has lagged behind that of many other prokaryotes, presenting the CRISPR-Cas technology an opportunity to resolve a long-existing issue. Here, we applied the Streptococcus pyogenes type II CRISPR-Cas9 (SpCRISPR-Cas9) system for genome editing in Clostridium acetobutylicum DSM792. We further explored the utility of the SpCRISPR-Cas9 machinery for gene-specific transcriptional repression. For proof-of-concept demonstration, a plasmid-encoded fluorescent protein gene was used for transcriptional repression in C. acetobutylicum Subsequently, we targeted the carbon catabolite repression (CCR) system of C. acetobutylicum through transcriptional repression of the hprK gene encoding HPr kinase/phosphorylase, leading to the coutilization of glucose and xylose, which are two abundant carbon sources from lignocellulosic feedstocks. Similar approaches based on SpCRISPR-Cas9 for genome editing and transcriptional repression were also demonstrated in Clostridium pasteurianum ATCC 6013. As such, this work lays a foundation for the derivation of clostridial strains for industrial purposes.

Importance: After recognizing the industrial potential of Clostridium for decades, methods for the genetic manipulation of these anaerobic bacteria are still underdeveloped. This study reports the implementation of CRISPR-Cas technology for genome editing and transcriptional regulation in Clostridium acetobutylicum, which is arguably the most common industrial clostridial strain. The developed genetic tools enable simpler, more reliable, and more extensive derivation of C. acetobutylicum mutant strains for industrial purposes. Similar approaches were also demonstrated in Clostridium pasteurianum, another clostridial strain that is capable of utilizing glycerol as the carbon source for butanol fermentation, and therefore can be arguably applied in other clostridial strains.

FIG 1

Genome editing in C. acetobutylicum using the type II CRISPR-Cas9 system of S. pyogenes. Expression of a plasmid-encoded Cas9 with a programmable sgRNA results in Cas9-mediated interference of the C. acetobutylicum chromosome and cell death. (A) Introduction of a double-stranded DNA break at the cac1502 locus was achieved by targeting a sgRNA, consisting of the Cas9 binding handle sequence (blue) and a spacer sequence complementary to the protospacer sequence present on the C. acetobutylicum chromosome (green). Cas9 is guided to the target locus by the sgRNA through base pairing of the spacer sequence to the chromosomal protospacer, and recognition of the S. pyogenes PAM element (5′-NGG-3′, orange) by Cas9 initiates strand separation of the host chromosome and endonucleolytic activity of Cas9 resulting in the introduction of a double-stranded DNA break. (B) Introduction of a cac1502 editing cassette to the CRISPR expression plasmid allows DCHR between the plasmid and the host chromosome. Two alternative DCHR events are shown, one introducing a deletion (pCas9gRNA-delcac824I) and one introducing a gene replacement with afp (pCas9gRNA-delcac8241-afp). Each of these events is expected to result in the deletion of the cac1502 coding sequence as well as the protospacer and PAM element required for recognition and cleavage by Cas9 and the establishment of transformation.

FIG 2

Confirmation of gene deletion and replacement using SpCRISPR-Cas in C. acetobutylicum. (A) Schematic depicting the genomic structures of the C. acetobutylicum wild type (DSM792), markerless deletion of cac1502 (792Δcac1502), and markerless gene replacement of cac1502 with P_thl::afp (792Δcac1502::afp) and the primers used for colony PCR verification of genome editing. (B) Colony PCR verification of gene editing (lane 1, unedited C. acetobutylicum genomic DNA; lanes 2 and 3, verification of genome editing using ∼500 bp and ∼1 kbp of the homologous region, respectively; lane 4, negative control for primers AFPcolpcr.f and cac1502colPCR.r using unedited C. acetobutylicum genomic DNA as the template; lanes 5 and 6, verification of P_thl::afp integration at the cac1502 locus). (C) Fluorescence intensity of the C. acetobutylicum wild-type and AFP-producing mutant via fluorescent plate reader.

FIG 3

CRISPRi-mediated transcriptional repression of afp in Clostridium. (A) Mean fluorescence intensity for DSM792 harboring plasmids pMTL85141, pGLOW-CK^XN-Pp1, pCRISPRi-Cont, pCRISPRi-AFP, and pCRISPRi-AFPT analyzed by fluorescent plate reader. (B) Histogram of fluorescence intensity of DSM792 harboring pCRISPRi-Cont, empty control vector pMTL85141, or pCRISPRi-AFP as analyzed by flow cytometry.

FIG 4

CRISPRi-mediated repression of HPrK leading to the coconsumption of glucose and xylose. Percent residual glucose and xylose and growth curve (inset) for DSM792 harboring plasmids pCRISPRi, pCRISPRi-GlpX, and pCRISPRi-HPrk.