TABLE 3.

Protocols used for plasmid curing/ARGs elimination via CRISPR.

Curing/Elimination strategy	Species of interest	Plasmid/Genes target	Delivery	Results	References
Plasmid conjugation blocking by CRISPR system via complementarity between spc1 CRISPR locus and a nes gene region from staphylococcal plasmids	S. aureus S. epidermidis	pG0400 plasmid nes gene	Conjugation Electroporation	Plasmid conjugation and transformation blocking Limiting the spread of antibiotic resistance	Marraffini and Sontheimer, 2008
S. pneumoniae: introducing the ermAM gene together with a premature stop codon in the srtA locus. E. coli pCRISPR:rpsL plasmid construct with a spacer that would guide Cas9 cleavage	S. pneumoniae E. coli	ermAM, erythromycin resistance gene rpsL, streptomycin resistance gene	Transformation	Killing of transformed cells CRISPR selection of non-edited cells	Jiang et al., 2013
CRISPR plasmid constructs bearing the copy of target genes, introduction of double-strand breaks by complementary RNA-guided nucleases in targets CRISPR bacteriophage constructs using for targeting plasmids of interest	E. coli Galleria mellonella	blaSHV–18 blaNDM–1 pZE-blaNDM–1–gfp	Conjugation Viral transduction	Sequence-specific cytotoxicity Excluding high-copy antibiotic resistance plasmids Re-sensitizing a resistant population to antibiotics.	Citorik et al., 2014
Insertion of CRISPR array in a staphylococcal vector to obtain pDB114, programmed to target kanamycin resistant gene Antimicrobial CRISPR cas phagemid to target the methicillin resistance gene CRISPR array that target plasmids of interest	S. aureus	aph-3, kanamycin resistance gene mecA, methicillin resistance gene pUSA01 pUSA02 pUSA03	Transformation Transduction	Sequence-specific killing of staphylococci resistant to kanamycin or methicillin Loss of pUSA02 plasmid Immunization of staphylococci against pUSA02 transfer.	Bikard et al., 2014
Phage transferable CRISPR cas system	E. coli	blaNDM–1 and blaCTX–M–15 encoding resistance to carbapenems pNDM and pCTX antibiotic resistance plasmid	Lysogenization Transformation	Resistance plasmid curing Prevention of horizontal gene transfer Sensitizing bacteria to multiple antibiotic resistance genes	Yosef et al., 2015
CRISPR plasmid construct, able to recognize target sequences from ESBL strains	E. coli	bla, beta-lactamase genes tet, tetracycline resistance gene pUC19 mediating Amp resistance pET21b pBR322	Transformation Conjugation	Re-sensitization to antibiotics of E. coli carrying ESBL plasmids CRISPR mediated clearance of whole plasmids	Kim et al., 2016
CRISPR plasmid designed to target a sequence from the replicase gene	Zymomonas mobilis	pZZM402 pZZM403	Transformation Electroporation	Elimination of native plasmid of Z. mobilis	Cao et al., 2017
Targeting conserved regions from colE1 replicons via CRISPR	E. coli P. putida	pZE-GFP, pZA-GFP pZS-GFP	Transformation	Efficient plasmid curing	Lauritsen et al., 2017
mcr-1 knockout via pCas: mcr CRISPR plasmid	E. coli	Plasmid-borne mcr-1 gene, colistin resistant.	Electroporation BMAP-27 antimicrobial peptide	Sensitization of E. coli strains to colistin following mcr-1 elimination	Sun et al., 2017
pLQ-Pxyl/tet-cas9-Pspac-sgRNA construct, designed to target plasmid of interest	S. aureus	pLQ-KO-tgt-50 bp pLQ-KO-rocA	Transformation	Efficient editing of the target locus	Liu et al., 2017
CRISPR -based plasmid pHCas9 targeting pHT01 and pB0A	B. subtilis	pHT01 pB0A	Transformation	Both plasmids cured by serial culture in antibiotic-free conditions	So et al., 2017
CRISPR2 locus manipulation to obtain pCR2-ermB and pCR2-Phage1	E. faecalis	PRP pTEF1 pAM771	Conjugation Electroporation	Decrease in conjugation frequency of the plasmids harboring ARGs	Hullahalli et al., 2017
CRISPR Cas 9 – based plasmid curing system (pFREE) targeting all major plasmid replicon in molecular biology	E. coli	SEVA vectors	Transformation	Efficient curing of target plasmids	Lauritsen et al., 2018
pMCas9- mcr-1 CRISPR construct able to eliminate mcr-1 gene pMob-Cas9 construct, delivered by conjugation	E. coli	Plasmid-borne mcr-1 gene, conferring colistin resistance	Transformation Conjugation	Elimination of plasmid-borne mcr-1 via CRISPR system delivered via transformation and conjugation assay	Dong et al., 2019
Metal stressors exposure	E. coli	pKJK5 broad range plasmid	Conjugation Filter mating experiments	Plasmid elimination	Klumper et al., 2017