CRISPR-Cas9 Genome-editing Vectors
The CRISPR Cas9 nuclease of Streptococcus pyogenes (Sp) is the basis of many prokaryotic genome editing systems. Its deployment relies on recombination-based replacement of a target sequence with the desired mutant allele and subsequent elimination of the progenitor cell population through RNA-guided, Cas9-mediated cleavage of the parental allele. Expression of the native SpCas9 in either the E. coli donor or target cell is not well tolerated. We have developed two Clostridium/E.coli shuttle vector CRISPR/Cas9 systems that minimise SpCas9 toxicity.
An 84 nucleotide, theophylline responsive riboswitch, which minimises cas9 expression in the absence of inducer. This allows homologous recombination to occur in the target Clostridium before Cas9 mediates the site-specific double strand break. After induction, cas9 is expressed at a level that is sufficient for effective genome DNA cleavage but which avoids the toxicity associated with Cas9 overexpression . The universal nature of RiboCas was demonstrated through its successful application in C. sporogenes, C. pasteurianum, C. difficile, C. botulinum , C. autoethanogenum  and C. butyricum . As riboswitches are RNA-based devices, RiboCas should function in bacterial chassis other than Clostridium and in a range of applications for which tight control of gene expression is required.
Supplied as a kit HERE, comprising five vectors (pRECas1, pRECas2, pRECas1-p15a, pRGCas1 & pRGCas2) that incorporate two different riboswitches controlling Cas9 (E&G), and a choice of promoters controlling the expression of the sgRNA, either ParaE (1) or the J23119 promoter (2). Four of the plasmids are based on the ColE1 replicon, while the fifth uses the low copy number p15a replication region.
(ii) trCas9 (truncated Cas9)
Many CRISPR-Cas9 systems suffer from toxicity issues in both E. coli and the intended host as a consequence of overproduction of Cas9. The use of a Cas9 nickase (nCas9), a Cas9 mutant that introduces a breakage on only one strand of the chromosome, is less toxic to the cell and has been shown to enable the isolation of Clostridium mutants . We have developed a system based on a spontaneous, frameshift mutant that results in a truncated Cas9 protein (trcCas9) lacking 87 amino acids encompassing a RuvCI nucleolytic domain and is believed to act as a nickase . The mutation allowed Sp cas9 to be cloned downstream of the strong Pthl promoter and may find application where the use of strong, constitutive promoters is preferred.
The five vectors (pMTL431521, pMTL431511, pMTL431211, pMTL432211, pMTL431541) that are supplied as part of the kit, HERE, are all based on the pCB102 Gram +ve replicon, rely on the Pthl promoter for trCas9 expression, carrying the oriT region of RK2, incorporate either ermB or catP as the selectable marker, carry ColE1 or p15a as the Gram -ve replicon and rely on either the ParaE or PJ23119 promoter for expression of the sgRNA.
 Cañadas IC, Groothuis D, Zygouropoulou M, Rodrigues R, Minton NP. RiboCas: A Universal CRISPR-Based Editing Tool for Clostridium. ACS Synth Biol. 2019; 8(6):1379-1390.
 Seys FM, Rowe P, Bolt EL, Humphreys CM, Minton NP. A Gold Standard, CRISPR/Cas9-Based Complementation Strategy Reliant on 24 Nucleotide Bookmark Sequences. Genes (Basel). 2020; 11(4):458.
 Andrew Dempster, unpublished data
 Li Q, Chen J, Minton NP, Zhang Y, Wen Z, Liu J, Yang H, Zeng Z, Ren X, Yang J, Gu Y, Jiang W, Jiang Y, Yang S. CRISPR-based genome editing and expression control systems in Clostridium acetobutylicum and Clostridium beijerinckii. Biotechnol J. 2016; 11(7):961-72.
 Ingle P, Groothuis D, Rowe P, Huang H, Cockayne A, Kuehne SA, Jiang W, Gu Y, Humphreys CM, Minton NP. Generation of a fully erythromycin-sensitive strain of Clostridioides difficile using a novel CRISPR-Cas9 genome editing system. Sci Rep. 2019; 9(1):8123.