eISSN 1738-8872
pISSN 1017-7825
pISSN 1017-7825
Table. 2.
Table. 2.
Application of CRISPR/Cas9-mediated engineering in
| Cas | Target | Strategy | Vector | Repair | Organism | Related secondary metabolite | Remark | Ref | |
|---|---|---|---|---|---|---|---|---|---|
| SpCas9 | actI-orf1 | Disruption | pCRISPR-Cas9 | NHEJ | ACT | Reconstituted NHEJ with ligD expression | [86] | ||
| actVB | Disruption | pCRISPR-Cas9 | NHEJ | ACT | Reconstituted NHEJ with ligD expression | ||||
| actI-orf2 | Disruption | pWHU | NHEJ | ACT | codA(sm)-based screening system for plasmid-cured strain | [88] | |||
| zwf2 | Disruption | pCRISPomyces | NHEJ | S. rimosus | - | Oxytetracycline production enhancement by disruption of competitive gene | [93] | ||
| devB | Disruption | pCRISPomyces | NHEJ | S. rimosus | - | Oxytetracycline production enhancement by disruption of competitive gene | |||
| sshg_00040 - sshg_00050 | Deletion | pCRISPomyces | HDR | |
Lanthipeptide | - | [50] | ||
| sshg_05713 | Deletion | pCRISPomyces | HDR | |
Polycylic tetramic acid macrolactam | - | |||
| Formicamycin cluster | Deletion | pCRISPomyces | HDR | S. formicae | Formicamycin | - | [91] | ||
| forV | Deletion | pCRISPomyces | HDR | S. formicae | Formicamycin | - | |||
| actVA-orf5 | Deletion | pCRISPomyces | HDR | S. lividans | ACT | - | [50] | ||
| redD | - | redF | Deletion | pCRISPomyces | HDR | S. lividans | RED | - | |
| redN | Deletion | pCRISPomyces | HDR | S. lividans | RED | - | |||
| actVA-orf5 and redN | Deletion | pCRISPomyces | HDR | S. lividans | ACT and RED | Multiplexed editing | |||
| devB | Deletion | pCRISPomyces | HDR | S. rimosus | - | Oxytetracycline production enhancement by disruption of competitive gene | [93] | ||
| zwf2 | Deletion | pCRISPomyces | HDR | S. rimosus | - | Oxytetracycline production enhancement by disruption of competitive gene | |||
| phpD | Deletion | pCRISPomyces | HDR | S. viridochromogenes | Phosphinothricin tripeptide | - | [50] | ||
| phpM | Deletion | pCRISPomyces | HDR | S. viridochromogenes | Phosphinothricin tripeptide | - | |||
| sceN | Deletion | pCRISPR-Cas9 | HDR | BGC11 (sceliphrolactam) | - | [18] | |||
| sceQ-sceR fusion | Deletion | pCRISPR-Cas9 | HDR | BGC11 (sceliphrolactam) | Fusion of sceQ and sce R by deleting stop codon of sceQ, intergenic region between sceQ and sceR, and start codon of sceR | ||||
| actI-orf1 | Deletion | pCRISPR-Cas9 | HDR | ACT | - | [86] | |||
| actVB | Deletion | pCRISPR-Cas9 | HDR | ACT | - | ||||
| SpCas9 | ACT cluster | Deletion | pKCCas9 | HDR | ACT | - | [42] | ||
| actII-orf4 | Deletion | pKCCas9 | HDR | ACT | - | ||||
| actII-orf4 and redD | Deletion | pKCCas9 | HDR | ACT and RED | Multiplexed editing | ||||
| CDA cluster | Deletion | pKCCas9 | HDR | CDA | - | ||||
| glnR | Deletion | pKCCas9 | HDR | - | - | ||||
| RED cluster | Deletion | pKCCas9 | HDR | RED | - | ||||
| redD | Deletion | pKCCas9 | HDR | RED | - | ||||
| papR3 | Deletion | pKCCas9 | HDR | S. pristinaespiralis | pristinamycin | - | [90] | ||
| snaE1 and snaE2 | Deletion | pKCCas9 | HDR | S. pristinaespiralis | pristinamycin | - | |||
| actI-orf2 | Deletion | pWHU | HDR | ACT | Development of codA(sm)-based selection system for screening plasmid-cured strain | [88] | |||
| rpsL | Point mutation | pKCCas9 | HDR | - | Lys88Glu mutation | [42] | |||
| ACT, CDA, CPK, RED deleted region | Replacement | pKCCas9 | HDR | - | ΦC31 attB integration | [89] | |||
| Non-target BGCs | Replacement | pKCCas9 | HDR | S. pristinaespiralis | BGC2, 3, 5, 13, and 15 | Non-target BGC replacement with ΦC31 attB or ΦBT1 attB site | |||
| indC-like indigoidine synthase | Insertion | pCRISPomyces | HDR | |
Indigoidine | KasO* promoter knock-in to activate silent BGCs | [87] | ||
| redD | Insertion | pCRISPomyces | HDR | S. lividans | RED | KasO* promoter knock-in to activate silent BGCs | |||
| actII-orf4 | Insertion | pCRISPomyces | HDR | S. lividans | ACT | KasO* promoter knock-in to activate silent BGCs | |||
| frbD operon and frbC homolog | Insertion | pCRISPomyces | HDR | S. roseosporus | FR-900098 | KasO* promoter knock-in to activate silent BGCs | |||
| main synthase gene | Insertion | pCRISPomyces | HDR | S. roseosporus | BGC3 (T1pks) | KasO* promoter knock-in to activate silent BGCs | |||
| luxR-type regulator | Insertion | pCRISPomyces | HDR | S. roseosporus | BGC18 (T1pks) | KasO* promoter knock-in to activate silent BGCs | |||
| SSGG_RS0133915 | Insertion | pCRISPomyces | HDR | S. roseosporus | BGC24 (Nrps-t1pks) | KasO* promoter knock-in to activate silent BGCs | |||
| rppA and cytochrome P450 | Insertion | pCRISPomyces | HDR | S. venezuelae | BGC16 (T3pks) | KasO* promoter knock-in to activate silent BGCs | |||
| SSQG_RS26895-RS26920 operon | Insertion | pCRISPomyces | HDR | S. viridochromogenes | BGC22 (T2pks) | KasO* promoter knock-in to activate silent BGCs | |||
| rkD | Cloning | - | - | - | RK-682 | ICE | [95] | ||
| homE | Cloning | - | - | - | Holomycin | ICE | |||
| stuE~stuF2 | Cloning | - | - | - | Tü 3010 | ICE | [98] | ||
| stuD1, stuD2 | Cloning | - | - | - | Tü 3010 | ICE | |||
| SpCas9 | Tetarimycin BGC | Cloning | - | - | - | Tetarimycin | mCRISTAR | [97] | |
| spr1 region (pglE - snbC) | Cloning | - | - | - | Pristinamycin | mCRISTAR | [90] | ||
| 5-oxomilbemycin BGC | Cloning | - | - | - | 5-oxomilbemycin | mCRISTAR | [99] | ||
| Jadomycin and chlortetracycline BGC | Cloning | - | - | - | Jadomycin, and chlortetracycline | CATCH | [96] | ||
| Chloramphenicol, YM-216391, and pristinamycin II BGCs | Cloning | - | - | - | Chloramphenicol, YM-216391, and pristinamycin | CRISPR/Cas9 cleavage and Gibson assembly | [89] | ||
| SpdCas9 | actI-orf1 | CRISPRi | pCRISPR-dCas9 | - | ACT | - | [86] | ||
| actI-orf1 | CRISPRi | pSET-dCas9 | - | ACT | - | [101] | |||
| actII-orf4 | CRISPRi | pSET-dCas9 | - | ACT | - | ||||
| cdaPS1 | CRISPRi | pSET-dCas9 | - | CDA | - | ||||
| cpkA | CRISPRi | pSET-dCas9 | - | CPK | - | ||||
| redQ | CRISPRi | pSET-dCas9 | - | RED | - | ||||
| actI-orf1 and cdaPS1 | CRISPRi | pSET-dCas9 | - | ACT, CDA | Multiplexed editing | ||||
| actI-orf1 and cdaPS1, cpkA | CRISPRi | pSET-dCas9 | - | ACT, CDA, and CPK | Multiplexed editing | ||||
| actI-orf1, cdaPS1, and cpkA, redQ | CRISPRi | pSET-dCas9 | - | ACT, RED, CDA, and CPK | Multiplexed editing | ||||
| Proteins with AmiR and NasR Transcriptional Antiterminator Regulator domain (ANTAR) | CRISPRi | pSET-dCas9 | - | - | Gene essentiality test | ||||
| FnCpf1 | actI-orf1 | Disruption | pKCCpf1 | NHEJ | ACT | - | [106] | ||
| actI-orf1 | Disruption | pKCCpf1 | NHEJ | ACT | Reconstituted NHEJ with ligD and Ku expression | ||||
| redX | Disruption | pKCCpf1 | NHEJ | RED | - | ||||
| redX | Disruption | pKCCpf1 | NHEJ | RED Reconstituted NHEJ with ligD and Ku expression | |||||
| redX, redG | Deletion | pKCCpf1 | NHEJ | RED | Deletion by reconstituted NHEJ with ligD and Ku expression at two cleavage sites | ||||
| actI-orfI | Deletion | pKCCpf1 | HDR | ACT | - | ||||
| redX | Deletion | pKCCpf1 | HDR | RED | - | ||||
| actI-orf1, redX | Deletion | pKCCpf1 | HDR | ACT and RED | Multiplexed editing | ||||
| SBI00792 | Deletion | pKCCpf1 | HDR | Adjacent to 5-oxomilbemycin | - | ||||
| FnddCpf1 | actI-orf1 | CRISPRi | pSETddCpf1 | - | ACT | - | |||
| redX | CRISPRi | pSETddCpf1 | - | RED | - | ||||
| cpkA | CRISPRi | pSETddCpf1 | - | CPK | - | ||||
| redX, actI-orf1, and cpkA | CRISPRi | pSETddCpf1 | - | RED, ACT, and CPK | Multiplexed editing |
J. Microbiol. Biotechnol. 2019;29:667~686 https://doi.org/10.4014/jmb.1904.04015
© J. Microbiol. Biotechnol.