TABLE 1.

CRISPR‐Cas‐based phage design for controlling bacteria.

Phage	Target organism		Genome engineering in phage	Genes targeted in host organism	In vivo model	Highlights	References
	Name	Description
ΦNM1	S. aureus RNK	Kanamycin‐resistant	Phage loaded with pDB121::aph, pDB121::mecA	aph‐3, mecA	CD‐1 mice	Antibiotic resensitization Growth inhibition Selective killing Prevention of skin infection in mice model	91
	S. aureus USA300	Methicillin‐ and tetracycline‐resistant
M13	E. coli EMG2 pNDM‐1	β‐Lactam resistant	Phage loaded with RGNndm‐1, RGNndm‐1/shv‐18, RGNeae	bla NDM‐1, bla SHV‐18, gyr AD87G, eae	G. mellonella	Growth inhibition Multiplexing against different genetic signatures Targeting point mutant protein with antibiotic‐ resistance Remodulating synthetic consortium	90
	E. coli pSHV‐18	β‐Lactam resistant
	E. coli O157:H7	Enterohemorrhagic strain
	E. coli gyr AD87G	Quinolone resistant
	E. coli CJ236	Chloramphenicol resistant
T7	E. coli DS7045	E. coli BL21A carrying/pWUR397/pWUR400/pAnti‐1.7	1.7 gene, 4.3 gene	NA	NA	A simple and efficient method to genetically engineer the E. coli phage T7 using E. coli type I‐E CRISPR‐Cas system.	59
	E. coli RK6617	E. coli BL21A carrying/pWUR397/pWUR400/pAnti‐4.3
λ prophage	E. coli pNDM, or pCTX	Streptomycin resistance	CRISPR cascade genes and array in phage genome a	ndm‐1, ctx‐M‐15	NA	A strategy to sensitize bacteria to antibiotics and selectively kill antibiotic resistant bacteria	92
φSaBov	S. aureus CTH96	Bovine isolate susceptible to ϕSaBov	Programmed CRISPR‐Cas9 system in noncoding region of phage genome b	nuc, esxA	C57BL/6 mice	Growth inhibition of S. aureus Complementation of tail fiber for phage broad host range Removal of toxic gene from host chromosomes Decolonization of S. aureus from skin of mouse model	148
	S. aureus ST1, ST5, ST8 and ST36	Human pandemic clonal lineage
phiKpS2	K. pneumoniae S2	Mutant of K. pneumoniae DSM 2026	Δgp1_8 and Δholin	NA	NA	A CRISPR driven strategy for precise genome engineering in phage	43
φSaBov	S. aureus ATCC 6538‐GFP	A human isolated S. aureus strain engineered to express GFP	Programmed CRISPR‐Cas9 system in noncoding region of phage genome b	nuc	Sprague Dawley female rats	Mitigating biofilm forming S. aureus infection In vivo rat model for osteomyelitis and soft tissue infection Delivery of phage or phage‐fosfomycin therapeutics with alginate hydrogel Phage reducing soft tissue infection but not bone infection	149
M13	E. coli NEB5‐α bla IMP‐1 /bla OXA‐48 /bla VIM‐2 /bla NDM‐1 /bla KPC‐2/mcr‐1/mcr‐2	Carbapenem‐or colistin‐resistant	EC‐CapsidCas13a_blaIMP‐1/1/blaOXA‐48/blaVIM‐2/blaNDM‐1/blaKPC‐2/mcr‐1/mcr‐2, SA‐CapsidCas13a_mecA, mcr‐1, mcr‐2	bla IMP‐1, bla OX48 , bla VIM‐2 , bla NDM‐1 , bla KPC‐2 , mecA	G. mellonella	Sequence‐specific bactericidal activity Sequence‐specific killing to modulate the complex microbial flora In vivo therapeutic effect in G. mellonella infection model	94
	S. aureus USA300	Methicillin‐resistant
M13	E. coli SmR F+ sfgfp	Streptomycin resistant, green fluorescence protein expression	Phage loaded with pCas9‐GFPT‐f1A/B	sfgfp	BALB/c mice	Oral administration of M19 phage loaded with CRISPR‐Cas9 expressing phagemid for targeting sequence specific gene in gut microbiome A proof of principle for in vivo targeting strain specific organisms in gut microbiome	93
	E. coli SmR F+ mcherry	Streptomycin resistant, red florescence protein expression
vB_EcoM‐IME365	E. coli MG1655 pUCtargetk	Kanamycin‐resistance	Phage genome integrated with CRISPR‐Cas9 cassette from pCas9 plasmid	bla NDM‐1	BALB/c mice	Phage‐delivered resistance eradication with subsequent antibiotic treatment (PRESA) strategy to combat drug resistant pathogen The designed strategy displayed superior antimicrobial activity compared to lytic phage alone in in vitro and in vivo mouse skin and intestinal infection model	62

^a CRISPR cascade genes: cse1, cse2, cas7, cas5, cas6e, and cas3 of E. coli type I‐E CRISPR system, and CRISPR array encoding spacers to targeting ndm‐1 and ctx‐M‐15.

^b CRISPR‐Cas9 system: tracrRNA, crRNA, and spCas9.

pDB91: a phagemid system containing rinA, ter, S and terL genes and packaging site of ΦNM1; pDB121::aph: pDB91 carrying spCas9, tracrRNA, and minimal CRISPR array to target aph‐3; Aph‐3: aminoglycoside phosphotransferase gene; mecA: penicillin binding protein 2a; bla _ndm‐1 : New Delhi metallo‐β‐lactamase‐1; bla _shv‐18 : β‐lactamase; gyr: gyrase; eae: intimin; 1.7: nucleotide kinase; 4.3: Gp4.3; ctx‐M‐15: Cefotaximase‐Munich; mcr‐1: probable phosphatidylethanolamine transferase; nuc: micrococcal nuclease; esxA: Type VII secretion system extracellular protein A; bla _IMP‐1: β‐lactamase; bla _OXA‐48 : β‐lactamase, bla _VIM‐I : β‐lactamase class B VIM‐2: bla _KPC‐2 : β‐lactamase; sfgfp: super folder green florescence protein; mecA; pDB121::mecA: pDB91 carrying spCas9, tracrRNA, and minimal CRISPR array to target mecA; RGN: a phagemid system containing f1 origin and RNA guided nuclease construct; RGNndm‐1: RGN targeting New‐Delhi metallo‐β‐lactamase 1 gene; RGNndm‐1/shv‐18: RGN targeting both ndm‐1 and shv‐18 genes; RGNeae: RGN targeting both eae genes; pWUR397: cas3 under T7 promoter, KanR; pWUR400: cascade genes under T7 promoter, StrR; pAnti‐1.7 pWUR477 cloned with anti‐1.7 spacer; pAnti‐4.3 pWUR477 cloned with anti‐4.3 spacer; pCas9‐GFPT‐f1A/B: GFP targeting CRISPR‐Cas9 phagemid with bla and f1 ori sequence; EC‐Capsid‐Cas13a_blaIMP‐1/1/blaOXA‐48/blaVIM‐2/blaNDM‐1/blaKPC‐2/mcr‐1/mcr‐2: Vector expressing Cas13a and spacers for targeting either bla _IMP‐1 /bla _OXA‐48 /bla _VIM‐2 /bla _NDM‐1 /bla _{KPC‐2/mcr‐1/mcr‐2} , respectively; SA‐CapsidCas13a_mecA: vector expressing Cas13a and spacer for targeting mecA in S. aureus _. pUCtarget_k: pUC plasmid containing kanamycin resistance marker with 5′‐end consisting PAM sequence from blaNDM.