Table 2.

Advantages and disadvantages of most commonly used genome-editing methodologies in bacteria.

Method	Advantages	Disadvantages
“Suicide” plasmids	-Low cost -Does not require specialized strains -Useful for large genomic deletions or targeted gene disruption	-Low efficiency -High rate of false positives -Often requires several rounds of antibiotic selection -Long homology flanking regions (~1 Kb) to the desired edit need to be cloned
“Recombineering” (Lambda Red, RecE/T)	-Low cost -Highly efficient, particularly for small-scale edits -Utilizes DNA templates with only short regions of homology (50 bp) to promote gene edition by homologous recombination	-Requires development of specialized strains with controlled foreign recombinase expression. -Usually requires counter-selection steps to eliminate antibiotic resistance markers from the genome
ClosTron method (Retrotransposition-Activated Marker)	-Can be programmed by designing a 344 bp region homologous to the target gene -Broad-host range of Ll.LtrB intron theoretically allows its use in any bacterial species	-So far only tested in members of the Clostridium (Clostridiodes) genus -Requires extensive cloning or expensive out-sourced synthesis of modified targeting intron -Application of the method is straightforward only for targeted gene disruption
CRISPR-Cas (plasmid-encoded)	-Low cost -Can be combined with recombineering for an enhanced efficiency -Highly customizable -Double strand breaks induce cell death in non-edited cells diminishing background (false positive colonies) -Highly versatile genome editing from large genome deletions/insertions to single base mutations.	-High cytotoxicity of Cas9 expression can alter morphology and survival even when devoid of nuclease activity due to steric hindrance posed by Cas9 PAM binding and subsequent DNA unwinding activity along the genome. -Induction of off-target effects (undesired genome edits) due to non-specific DNA cleaving, particularly after prolonged Cas9/gRNA expression
CRISPR-Cas (Endogenous systems)	-Do not necessitate the expression of a foreign CRISPR nuclease -Highly programmable by altering the homology repair template and the CRISPR array sequence	-Requires extensive characterization of the endogenous CRISPR system (nucleases, PAM requirement, efficiency, etc.) and DNA repair pathways (e.g., NHEJ) for each particular species/strain