Table 3.

Tools for genome engineering of clostridia

Purpose	Tool	Description	Application	References
Genomic integration of whole pathways	Phage serine integrase system for dual integrase casette exchange (DICE)	Allows integrase-mediated site-specific integration into the genome without integration of unwanted DNA-like plasmid backbones	The whole butyric acid production pathway was integrated into the C. ljungdahlii genome	[112]
Genomic integration of whole pathways	Genomic integration system based on the Himar1 transposase	The Himar1 transposase is used to integrate the target DNA casette randomly at any AT-site in the genome	The acetone production pathway and an ermC selectable marker were integrated into the C. ljungdahlii genome	[220]
Deletion of single genes	CRISPR nickase based system for deletion	The truncated Cas9 protein (trCas9) lacking the RuvCl nucleolytic domain can be used for deletions even when expressed strong and constitutively	Two ermB genes and pyrE were deleted from the Clostridioides difficile genome	[118]
Deletion and integration of pathways	Targetron-recombinase system for large-scale genome engineering	Targetrons are used to position markerless lox66 and lox71 sites in the genome. Cre recombinase deletes the DNA in between the lox66 and lox71 site via homologous recombination	A 50-gene prophage island was deleted from the C. phytofermentans genome	[35]
Complementation after deletion	CRISPR/Cas9-based complementation strategy employing 24 nt bookmark sequences	A 24 nt bookmark sequence is introduced at the place of a gene that has been deleted. For future complementation studies, the 24 nt bookmark sequence is selected against to integrate the wildtype gene at its original location	The pyrE gene in C. ljungdahlii was replaced with 9 consecutive bookmark sequences. All 9 bookmark sequences allowed complementation with the pyrE wildtype gene	[264]
Editing of single nucleotides in genome	CRISPR-targeted base editing via deamination	A combination of nuclease deactivated Cas9 with activation-induced cytidine deaminase is applied for cytosine to thymine substitution without DNA cleavage	Premature stop codons were introduced into genes related to the formation of acetate (pta) and ethanol (adhE1, adhE2, aor1, aor2) in C. ljungdahlii	[320]
Editing of single nucleotides in genome	CRISPR nickase assisted base editing via deamination	A fusion of cytidine deaminase, CRISPR-Cas9D10A nickase and uracil DNA glycosylase inhibitor (UGI) is used for base-pair substitutions of C∙G to A∙T	Mutations were introduced into the pyrE, xylR, Spo0A and araR gene of C. beijerinckii	[163]