. 2022 Apr 9;11(4):571. doi: 10.3390/biology11040571

Table 2.

The Characteristics and Applications of BEs in Nonmodel Microbes.

Species	Major Function	Type	Year	gRNA Promoter	Construct of Fusion Protein	PAM	Editing Window	Editing Efficiency	Multiplex Gene Editing	Applications of BEs	Off-Targets	Refs
Industrially Important Microbes
Kluyveromyces marxianus	Industrial production of various enzymes, chemicals, and macromolecules, as well as the utilization of cell biomass	CBE	2017	P_SNR52	PTSNR52	NGG	−17 to −18	12.5–25%	nr	Inactivate Nej1 and Dnl4 to build NHEJ null mutants with an increased efficiency of homologous recombination and to facilitate multiple integration mediated by CRISPR/Cas9	nr	[69]
Psedomonas spp.	An excellent bacterial host to produce polymers, bulk chemicals, drugs, and high-price specialties	CBE	2018	P_trc	P_rpsL-rAPOBEC1-nCas9^D10A	NGG	−13 to −18	100%	nr	Inactivate genes in P. aeruginosa PAO1, P. putida KT2440, P. fluorescens GcM5-1A, and P. syringae DC3000 to test editing window and efficiency	nd in the six similar spacers of the rhlR and rhlB genes	[41]
		CBE	2020	P_j23119	P_bs/P_araBAD-rAPOBEC1-nCas9^D10A	na, none of the selected colonies achieved C-to-T mutations						[70]
		CBE	2020	P_j23119	P_bs-rAPOBEC1-eSpCas9pp^D10A
		CBE	2020	P_j23119	P_araBAD-rAPOBEC1-eSpCas9pp^D10A	NGG	nr	20%	nr	Edit ttgA to test editing efficiency	nr
		CBE	2020	P_j23119	P_m-rAPOBEC1-eSpCas9pp^D10A-UGI	NGG	−13 to −18	40–60%	nr	Edit ttgA to test editing efficiency	nr
		CBE	2020	P_j23119	P_araBAD-rAPOBEC1-eSpCas9pp^D10A-UGI	NGG	−13 to −18	80–100%	100% for double targets and 35% for triple targets	Inactivate genes in P. putida, P. aeruginosa, P. fluorescens, and P. entomophila to prove CBE general availability; simultaneously edit genes by one-plasmid and two-plasmid system	nd by Sanger sequencing the potential sites predicted by CasOT
		CBE	2020	P_j23119	P_araBAD-rAPOBEC1-eSpCas9pp^D10A-NG-UGI	NG	−13 to −18	100%	100% for double targets recognized by eSpCas9pp and eSpCas9-NG in a two-plasmid system	Inactivate pykA and pcaH in one step; mutate G136 in AroF-2 to select a mutant strain with increased PCA titer up to 264.87 mg/L	nr
		CBE	2020	P_j23119	P_araBAD-YE1-eSpCas9pp^D10A-UGI	NGG	−14 to −17	62.5%	nr	Precisely edit ttgA, which contains multiple cytidines with enhanced editing efficiency from 25% to 62.5%	nr
Yarrowia lipolytica	GRAS and industrial production of lipase and organic acids	CBE	2019	P_SCR1’-tRNA^Gly	P_{UAS1B8-TEF(136)}-nCas9^D10A-PmCDA1-UGI	NGG	−14 to −20	28%	6.7% for double targets	Inactivate TRP1, PEX10, HIS3 in ku70Δ strain to test editing efficiency	nr	[26]
Yarrowia lipolytica	GRAS and industrial production of lipase and organic acids	CBE	2019	P_SCR1’-tRNA^Gly	P_TEFin-nCas9^D10A-PmCDA1-UGI	NGG	−14 to −20	94.3 ± 5%	31% for double targets		nr	[26]
Streptomyces spp.	Industrial production of bioactive secondary metabolites, such as antifungals, antivirals, antitumorals, anti-hypertensives, and mainly antibiotics, etc.	CBE	2019	P_ermE*	P_tipA-rAPOBEC1-nCas9^D10A-UGI	NGG	−11 to −17	30–100%	33.3% for triple targets	Substitute amino acids in SCO5087 and SCO5092; inactivate genes of BGCs in nonmodel strain S. griseofuscus; efficiently and simultaneously inactivate two identical copies of kirN	38–56 by WGS (24–34 meaningful amino acid changes); whereas 29 SNVs in wild-type strain (18 amino acid changes);	[44]
		ABE	2019	P_ermE*	P_tipA-TadA-TadA*-nCas9^D10A-UGI	NGG	−12 to −17	0–100%	nr	Target SCO5087 and a designed matrix containing NA motifs to test efficiency and preference	27–33 by WGS (20–21 meaningful amino acid changes)	[44]
		CBE	2019	P_kasO*	P_rpsL-rAPOBEC1-dCas9-UGI	NGG	−13 to −17	43–70%	43% for double targets	Edit redD and actl to test C-to-T efficiency with a few C-to-G and C-to-A mutations	nr	[45]
		CBE	2019	P_kasO*	P_rpsL-rAPOBEC1-nCas9^D10A-UGI	NGG	−13 to −17	100%	100% for double targets; 60% for quintuple targets	Simultaneously disrupt the genes of polyketide synthase clusters to increase production of avermectin	3 by Sanger sequencing the sites predicted by CasOT;
		CBE	2019	P_kasO*	P_rpsL-rAPOBEC1-HF-nCas9^D10A-UGI	NGG	−13 to −17	80%	nr	Edit olm to test off-target events, which was decreased to an undetectable level	nd by Sanger sequencing the sites mentioned above
		ABE	2019	P_kasO*	P_rpsL-TadA-TadA-dCas9*	na, all selected colonies showed the A/G overlapping peak in sanger sequencing
		ABE	2019	P_kasO*	P_rpsL-TadA-TadA*-nCas9^D10A	NGG	−14 to −17	100%	nr	Disrupt the initiation of actVB translation by converting ATG start codon to ACG to accumulate actinoperylone	nr
		CBE	2019	P_j23119	P_ermEp-dCas9-PmCDA1-UL*	na, growth of cells is severely delayed when CBE was overexpressed by the strong constitutive promoter						[46]
		CBE	2019	P_j23119	P_tipAp-dCas9-PmCDA1-UL	NGG	−16 to 20	10–100%	60% for double targets; 20% for triple targets	Inactivate genes in S. coelicolor and S.rapamycinicus to test editing efficiency and general availability to other strains	1 by Sanger sequencing the potential sites predicted by Cas-OFFinder
		CBE	2019	P_j23119	P_tipAp-nCas9^D10A-PmCDA1-UL	NGG	−16 to 20	15%	nr	Edit redW with low efficiency from C to T but 85% efficiency for C-to-G mutation	nr
		CBE	2021	P_{gapdh (EL)}	P_rpsL(XC)-rAPOBEC1-dCas9-UGI	NGG	−13 to −18	1–20%	nr	Edit redN, redD, and act_β-ketoacyl to test editing efficiency	16.50 ± 8.35 by WGS	[71]
		CBE	2021	P_{gapdh (EL)}	P_rpsL(XC)-rAPOBEC1-nCas9^D10A-UGI	NGG	−13 to −18	3–25%	nr		nr
		CBE	2021	P_{gapdh (EL)}	P_rpsL(XC)-rAPOBEC1-dCas9-UGI with asRNA	NGG	−13 to −18	21.2–65.8%	nr		13.50 ± 3.32 by WGS
		CBE	2021	P_{gapdh (EL)}	P_rpsL(XC)-rAPOBEC1-nCas9^D10A-UGI with asRNA	NGG	−13 to −18	26.2–79.4%	nr		nr
Clostridium beijerinckii	Production of acetone, n-butanol, isopropanol etc.	CBE	2019	P_j23119	P_thl-rAPOBEC1-nCas9^D10A-UGI	NGG	−13 to −17	20–100%	nr	Edit pyrE, xylR, spo0A, and araR to test efficiency of codon-optimized CBE; inactivate xylR to enhance the xylose fermentation	nr	[47]
Clostridium ljungdahlii	Production of acetic acid and ethanol from waste gas	CBE	2020	P_j23119	P_2TetO1-dCas9-PmCDA1-UL	NGG	−11 to −19	2–55.6%	nr	Inactivate adhE1 and aor2 separately to increase acetate yield as well as lower ethanol production under either heterotrophic or autotrophic conditions	nr	[72]
Rhodobacter sphaeroides	Industrial production of CoQ10, isoprenoids, poly-β-hydroxybutyrate, hydrogen	CBE	2020	P_j23119	P_Lac-dCas9-PmCDA1-UL	NGG	−14 to 20	16.7%	nr	Inactivate appA and ppsR to test efficiency with pure C-to-T conversion	nr	[48]
		CBE	2020	P_j23119	P_Lac-nCas9^D10A-PmCDA1-UL	NGG	14 to 20	10–96.7%	43% for double targets; 46.7% for triple targets	Inactivate appA, etc., to test C-to-T efficiency with C-to-G and C-to-A byproducts; disrupt ubiF, ubiA, ubiG, and ubiX to reveal their importance in the CoQ10 biosynthetic pathway	nr
		ABE	2020	P_j23119	P_Lac-TadA-TadA*-dCas9	NGG	−14 to −16	0–5%	nr	Edit appA, ppsR, crtB, and bchG to alter translation level or block translation initiation	nr
		ABE	2020	P_j23119	P_Lac-TadA-TadA*-nCas9^D10A	NGG	−14 to −16	0–30%	nr	Edit appA, etc to alter translation level or block translation initiation	nr
Shewanella oneidensis	Bioelectricity production from biomass wastes	CBE	2020	P_tac	P_rpsL-rAPOBEC1-nCas9^D10A	NGG	−13 to −18	33.3–100%	87.5% for double targets	Target NC motifs to test editing preference; inactivate gfp, blaA, and dmsE to test editing activity; identify key genes in GlcNAc or glucose metabolism to obtain a mutant strain with enhanced degradation efficiencies for organic pollutants	nr	[49]
Companilactobacillus crustorum	Production of bacteriocin and 3-phenyllactic acid	CBE	2021	P₃	P_sppA-rAPOBEC1-nCas9^D10A	NGG	−14 to −18	75–100%	nr	Edit seven C-rich spacer sequences in a plasmid to test editing window and efficiency	nr	[73]
Agriculturally Important Microbes
Paenibacillus polymyxa	Nitrogen fixation, plant growth promotion, soil phosphorus solubilization and production of cxopolysaccharides, hydrolytic enzymes, antibiotics, and cytokinin	CBE	2021	P_ara	P_grac-nCas9^D10A-PmCDA1	na, no transformant was obtained due to the toxicity of the fusion protein						[56]
Paenibacillus polymyxa		CBE	2021	P_ara	P_spac-dCas9-PmCDA1-UGI	NGG	−16 to 20	100%	100% for double and triple targets; 83.3% for quadruple targets; 75.5% for quintuple targets	Disrupt genes of five known BGCs to reveal the antimicrobial spectrum of the novel antibiotics in the sixth unknown BGCs	8.5 SNVs including 4.2 amino acid changes by WGS	[56]
Agrobacterium spp.	Nature’s genetic engineer for diverse species including crops	CBE	2021	P_j23119	P_aadA-dCas9-PmCDA1-UGI-LVA	na, no correct clones were obtained in E. coli* probably due to the toxicity*						[74]
Agrobacterium spp.		CBE	2021	P_j23119	P_virB-dCas9-PmCDA1-UL	NGG	−15 to −19	91%	80% for double targets	Inactivate recA to maintain stability for plant transformation; separately inactivate rolB, rolC, and orf13 to confirm their importance in hair root construction	nr	[74]
Sinorhizobium meliloti	Perform symbiotic nitrogen fixation within leguminous host plants such as alfalfa, an important forage crop	ABE	2021	P_SigA/P_RpoN/P_tyr	P_HemA-TadA-TadA-nCas9^D10A*	na, failed to mediate the A-to-G transition when gRNA is expressed by promoter SigA, RpoN or tyr						[75]
		ABE	2021	P_RpsT	P_HemA-TadA-TadA*-nCas9^D10A	NGG	−11 to −17	60%	nr	Edit nodA to test the editing efficiency	nr
		ABE	2021	P_RpmJ	P_HemA-TadA-TadA*-nCas9^D10A	NGG	−11 to −17	100%	90% for triple targets	Edit nodA, nodB, nodC, nifD, nifH, and nifK to test if the promoters can drive the expression of the fusion protein to perform efficient editing	nd by Sanger sequencing the potential sites predicted by Cas-OFFinder
		ABE	2021	P_RpmJ	P_Neo-TadA-TadA*-nCas9^D10A	NGG	−11 to −17	100%
		ABE	2021	P_RpmJ	P_Tau-TadA-TadA*-nCas9^D10A	NGG	−11 to −17	80%
		CBE	2021	P_RpmJ	P_HemA-rAPOBEC1-nCas9^D10A-UGI	NGG	−13 to −17	75%	nr	Inactivate nodA (W7*) to test if the growth of plants inoculated with the mutant strain was retarded
		CBE	2021	P_RpmJ	P_Tau-rAPOBEC1-nCas9^D10A-UGI	NGG	−13 to −17	100%	nr
		CBE	2021	P_RpmJ	P_HemA-nCas9^D10A-PmCDA1-UGI	NGG	−13 to −20	100%	80% for double targets; 50–70% for triple targets	Edit nodA, etc to test editing efficiency
		GBE	2021	P_RpmJ	P_HemA-nCas9^D10A-PmCDA1-UNG	NGG	−14 to −18	33–80%	nr	Edit nodA, etc to test editing efficiency	nr
Clinically Important Microbes
Brucella melitensis	The most important agent of human brucellosis	CBE	2018	P_LlacO-1	P_trc-rAPOBEC1-nCas9^D10A-UGI-NLS	NGG	−15	100%	nr	Inactivate virB10 by targeting three sites with 100% editing efficiency at only one site	nr	[37]
Klebsiella pneumoniae	Cause pneumonia, bloodstream infections, wound, or surgical site infections and meningitis; biosynthesize 1,3-propanediol and 2,3-butanediol	CBE	2018	P_j23119	P_rpsL-rAPOBEC1-nCas9^D10A	NGG	−13 to −18	100%	nr	Edit fosA and dhaK to test editing efficiency with a few C-to-A byproducts; inactivate the bla_KPC-2 and bla_CTX-M-65 to dissect drug-resistance mechanisms	nr	[60]
Staphylococcus aureus	Cause infections ranging from skin infections to severe systemic infections	CBE	2018	P_{cap 1A}	P_rpsL-rAPOBEC1-nCas9^D10A	NGG	−13 to −17	100%	nr	Inactivate agrA and cntA to test efficiency	nr	[76]
Staphylococcus aureus		ABE	2020	P_{cap 1A}	P_rpsL-ecTadA-TadA*-nCas9^D10A	NGG	−13 to −17	50–100%	100% for double targets	Screen key residues of cntBC targeted by 38 gRNAs to obtain 42 mutant strains	nd gRNA-dependent off-target by WGS	[77]
Acinetobacter baumannii	causing ventilator-associated pneumonia and bloodstream infections, and mortality rates can reach 35%	CBE	2019	P_j23119	Ptac-rAPOBEC1-nCas9^D10A	NGG	−13 to −18	20–100%	nr	Edit tynA, acel, adeB, cpdA, entE, and oxyR to test editing efficiency and preference of TC motifs; disrupt drug-resistance relevant genes blaOXA-23, blaTEM-1D, and blaADC-25 to dissect drug-resistance mechanisms	nr	[78]
Mycobacterium spp.	Causes tuberculosis, getting 10 million infections and 1.45 million deaths in 2018 worldwide	CBE	2021	P_j23119	P_tetR-rAPOBEC1-dSt1Cas9	NNRGAA	nr	4–15%	nr	Test availability of dSt1Cas9-BE in Mycobacterium with low efficiency for C-to-T but 18–70% efficiency for C-to-G	nr	[79]
		CBE	2021	P_j23119	P_tetR-rAPOBEC1-dSt1Cas9-UGI-UGI	NNRGAA	nr	12–95%	nr	Inactivate katG to obtain mutant strain with increasing resistance to Isoniazid treatment	nr
		CBE	2021	P_j23119	P_tetR-rAPOBEC1-dSt1Cas9_evolve-UGI-UGI	NNNNAA	−10 to −14	20–95%	87.5% for both double and triple targets	Inactivate the essential L-leucine biosynthesis genes leuB and lueC; inactivate ctpE to increase bacterium aggregation in the presence of EGTA	nd gRNA-dependent off-target by WGS
		GBE	2021	P_j23119	P_tetR-rAPOBEC1-dSt1Cas9-UNG	NNRGAA	nr	100%	nr	Edit five different loci to test editing efficiency	nr
		GBE	2021	P_j23119	P_tetR-rAPOBEC1-dSt1Cas9_evolve-UNG	NNNNAA	−13 to −16	20–65%	75% for triple targets	Edit 29 endogenous genomics sites to find only TC motif is available for editing	nr
		CBE	2021	P_j23119	P_tetO-rAPOBEC1-dSt1Cas9-UGI	NNAGGAC	nr	1.2%	nr	Inactivate gfp to test editing efficiency	nr	[80]
		CBE	2021	P_j23119	P_tetO-rAPOBEC1-nSt1Cas9-UGI	NNAGGAC	nr	10.3%	nr		nr
		CBE	2021	P_j23119	P_tetO-rAPOBEC1-nSt1Cas9-UGI with assistant plasmid expressing recX	NNAGGAC	−12 to −18	37.5–100%	nr		nr
		CBE	2021	P_j23119	P_tetO-rAPOBEC1-nSt1Cas9-UGI with assistant plasmid expressing recX and nucS_E107A	NNAGGAC	nr	12.5–75%	nr	Inavtivate Rv0582, Rv0627 and Rv2530 to test efficiency; Inactivate katG to build a mutant stain with higher 50% minimum inhibitory concentration than the wild-type strain	nr

nr: not reported, nd: not detected, na: not available, UL: UGI-LVA (protein degradation tag), SNVs: single-nucleotide variants, WGS: whole-genome sequencing, GRAS: generally recognized as safe, BGC: biosynthetic gene cluster. The construct of BEs that failed to work in microbes are marked in red.