Table 1.
Examples of small molecules that control the activity and duration of Cas nucleases and gRNAs/crRNAs.
| Small-molecule control of Cas9 expression levels | |||||||
| Cas nuclease | RNA | Small molecule | Mode of action | Note | Refs. | ||
| SpCas9, SaCas9, AsCas12a, RfxCas13d, base editor, prime editor |
gRNA, crRNA, pegRNA | Doxycycline | Doxycycline-induced synthesis of mRNAs encoding the genome editors. | Extra expression of rtTA is required. Currently used in a wide assortment of biological systems in vitro and in vivo. Enhancement of the genome-editing specificity was demonstrated. | [25,26,27,28,29,30,31,32,33,34] | ||
| SpCas9 | gRNA | Lys(Boc), an unnatural amino acid | Unnatural amino acid induces amber codon (UAG) suppression to generate full-length SpCas9. | Extra expression of aaRS and tRNA is required. | [35] | ||
| SpCas9, CjCas9 |
gRNA | G418 | G418 induces stop codon (UGA) read-through to generate full-length Cas9. | Could affect other cellular pathways. | [36] | ||
| SpCas9, base editor, prime editor |
gRNA, pegRNA | KPT330 | KPT330 inhibits the export of mRNAs encoding the genome editors. | Could affect other cellular pathways. Enhancement of the genome-editing specificity was demonstrated. | [38] | ||
| Small-molecule control of gRNA expression levels | |||||||
| Cas nuclease | RNA | Small molecule | Mode of action | Note | Ref. | ||
| SpCas9, SaCas9 |
gRNA | Doxycycline | Doxycycline-bound TetR dissociates from H1/TetO promoter, and gRNA transcription is initiated. | Extra expression of TetR is required. | [39,40,41] | ||
| SpCas9 | gRNA | Doxycycline | Doxycycline-induced production of a gRNA targeting the SpCas9 gene. | Extra expression of TetR is required. Irreversible system; SpCas9 gene is disrupted. Enhancement of the genome-editing specificity was demonstrated. | [42] | ||
| SpCas9 | gRNAs harboring LoxP sequences | 4HT | Activation of Cre-ERT2 by 4HT leads to the recombination of gRNA-encoding genes, thus promoting or blocking the gRNA synthesis. | Extra expression of Cre-ERT2 is required. Enhancement of the genome-editing specificity was demonstrated. | [43] | ||
| Small-molecule control of unmodified Cas nucleases | |||||||
| Cas nuclease | RNA | Small molecule | Mode of action | Note | Ref. | ||
| SpCas9, dSpCas9, base editor |
gRNA | BRD0539, BRD20322, BRD7087 | Inhibition of SpCas9−PAM interaction. | The first small-molecule SpCas9 inhibitor having potent cellular activities. Inhibitors of the various Cas9-based technologies. Sub-optimal efficacy and potency. | [21] | ||
| SpCas9 | gRNA | Compound 85 and analogs | Inhibition of gRNA loading on SpCas9. | Inhibits SpCas9 in E. coli. | [45] | ||
| SpCas9 | gRNA | 6 compounds from UCLA Molecular Shared Screening Resource | Inhibition of SpCas9 with unknown mechanism. | Several hit compounds inhibit SpCas9 in test tubes, but their high toxicity restricted cellular tests. | [46] | ||
| SpCas9 | gRNA | Valproic acid | Binds to SpCas9 to induce its thermal destabilization. | Photothermal triggers are required for efficient denaturation of SpCas9. | [47] | ||
| Small-molecule control of engineered Cas nucleases | |||||||
| Cas nuclease | RNA | Small molecule | Mode of action | Note | Ref. | ||
| AID-dSpCas9, AID-dSpCas9-PR, AID-dSaCas9-VP64 | gRNA | Auxin | Auxin-induced degradation of Cas9 proteins by the proteasome. | Rapid degradation of Cas9 proteins. Extra delivery of plant factors for ubiquitination is required. | [52] | ||
| SpCas9-FKBPF36V | gRNA | dTAG-47 | dTAG-47-induced degradation of SpCas9-FKBPF36V by the proteasome. | Enhancement of the genome-editing specificity was demonstrated. DNA repair outcome was altered by modulating SpCas9′s half-life. | [55] | ||
| SpCas9-FCPF, dSpCas9-FCPF, PdCas12a-FCPF, LwCas13a-FCPF, | gRNA, crRNA | A conjugate of lenalidomide and perfluoroaromatic moiety | Conjugate-induced degradation of Cas proteins by the proteasome. | Applicable to diverse Cas nucleases. | [57] | ||
| SpCas9-DHFR, SpCas9-ER50 | gRNA | TMP, 4HT | SpCas9-destabilizing domain fusions are stabilized by binding to small molecules. | Enhancement of the genome-editing specificity was demonstrated. Demonstrated in gene-drive applications. | [58,59] | ||
| SpCas9-DHFR | gRNA | Caged TMP molecules | SpCas9-DHFR fusion is stabilized by binding to TMP. | Spatiotemporal control was achieved using caged TMPs that release active TMP upon stimulation by reactive oxygen species or light. Demonstrated in vivo. | [60,61] | ||
| DD-SpCas9, DD-dSpCas9-VPR | gRNA | Shield-1 | DD-Cas9 fusions are stabilized by binding to Shield-1. | Demonstrated in vivo. | [62,63,64] | ||
| SpCas9 fused to a 4HT-responsive intein | gRNA | 4HT | 4HT binding to the intein initiates the protein splicing to release active SpCas9. | Enhancement of the genome-editing specificity was demonstrated. | [65] | ||
| SpCas9, dSpCas9-VPR, LbCas12a, AsCas12a, dLbCas12a-p65-HSF1 | gRNA, crRNA | Rapamycin | Rapamycin induces functional assembly of split Cas nucleases. | Demonstrated in vivo. | [67,68,69,70] | ||
| Base editors | gRNA | Rapamycin | Rapamycin induces functional assembly of split deaminases. | Genome-wide off-target base exchanges arising from the constitutively active deaminase were decreased. | [71,72] | ||
| SpCas9-ERT2, AsCas12a-ERT2 | gRNA, crRNA | 4HT | The binding of 4HT displaces the ERT2-bound cytoplasmic Hsp90 to localize the Cas-ERT2 fusions to the nucleus. | Enhancement of the genome-editing specificity was demonstrated. | [68,73,74] | ||
| BCL-xL and BH3 fused with SpCas9, dSpCas9-VPR, base editors, or prime editors | gRNA, pegRNA | A-385358, A1155463, WHEI-539 |
Autoinhibitory BCL-xL–BH3 interaction is inhibited by small molecules and Cas9 activity is restored. | Enhancement of the genome-editing specificity was demonstrated. Demonstrated with diverse dCas9 or nCas9-based technologies. | [75,76,77] | ||
| SpCas9 K866OABK mutant | gRNA | 2DPBA, 2DPBM | 2DPBA and 2DPBM are reacted with OABK to release functional lysine. | Extra expression of aaRS and tRNA is required when DNA is delivered. Appropriate for RNP delivery. | [79] | ||
| Base editor K1200TCOK mutant at nCas9 domain | gRNA | Me2Tz | Me2Tz is reacted with OABK to release functional lysine. | Extra expression of aaRS and tRNA is required when DNA is delivered. Appropriate for RNP delivery. | [80] | ||
| Small-molecule control of engineered gRNAs | |||||||
| Cas nuclease | RNA | Small molecule | Mode of action | Notes | Ref. | ||
| SpCas9, dSpCas9 | gRNA fused with an aptamer at the loop | Theophylline | Theophylline binding activates or deactivates gRNAs. | Demonstrated in E. coli for Cas9 nuclease activity and dCas9-based transcription modulation, and in human cells for dCas9-based transcription modulation. | [83,85,86] | ||
| SpCas9, dSpCas9-VPR | gRNA fused with an aptamer and a blocking motif at the 3′ end | Theophylline | Theophylline binding activates gRNAs. | Demonstrated in human cells for controlling the Cas9 nuclease activity. | [87] | ||
| SpCas9, dSpCas9-VPR, base editor | gRNA fused with an aptazyme at the 5′ end | Theophylline, guanine | Theophylline binding induces the self-cleavage by the aptazyme to release functional gRNAs. | Demonstrated in human cells for diverse Cas9-based technologies. | [88] | ||
| SpCas9 | gRNA containing C-to-G mutations at stem-loops | Naphthyridine carbamate dimer (NCD) | NCD binding deactivates gRNAs and switches off genome editing. | Demonstrated in human cells for inhibiting Cas9 nuclease activity. | [89] | ||
| SpCas9, LbCas13a, dLbCas13a, LbCas12a | gRNA or crRNA chemically masked by AMN groups | 2DPBM, TPPMS, THPP, TCEP | Phosphine compounds react with the AMN group to unmask and activate gRNAs/crRNAs | Demonstrated in human cells for various Cas proteins. | [90,91,92] | ||
| SpCas9, LbCas13a | Adamantoylated gRNA or crRNA | CB7 | Host–guest complexation increased the bulkiness of gRNAs/crRNAs to inactivate them. | Demonstrated in human cells for inhibiting Cas9 nuclease activity. | [93] | ||
| SpCas9, LbCas13a | Azido-group-containing gRNA or crRNA | DBCO-containing molecule | Click chemistry increased the bulkiness of gRNAs/crRNAs to inactivate them. | Demonstrated in human cells for inhibiting Cas9 nuclease activity. | [94] | ||
| Small-molecule control of anti-CRISPR proteins | |||||||
| Cas nuclease | RNA | Acr protein | Small molecule | Mode of action | Notes | Ref. | |
| SpCas9 | gRNA | AcrIIA4-DHFR | TMP | AcrIIA4-DHFR fusion is stabilized by TMP, and SpCas9 is inhibited. | Enhancement of the genome-editing specificity was demonstrated. | [96] | |
| dSpCas9-VPR | gRNA | DD-AcrIIA4 | Shield-1 | DD-AcrIIA4 fusion is stabilized by Shield-1, and SpCas9 is inhibited. | Shield-1 dose-dependent inhibition of gene expression was demonstrated. | [97] | |
| SpCas9, St3Cas9, prime editor |
gRNA, pegRNA |
AcrIIA25.1 or AcrIIA32.1 fused to a 4-HT-dependent intein | 4HT | Acr proteins fused to a ligand-responsive intein are activated by binding to 4HT followed by protein splicing. | 4HT-dependent inhibition of various Cas proteins was demonstrated. | [98] | |
| dSpCas9 | gRNA | AcrIIA4-hER-HBD | β-estradiol | AcrIIA4-hER-HBD is translocated to the nucleus by binding to β-estradiol. | Control of the dSpCas9-based gene expression was demonstrated in yeast. | [99] | |
| dSpCas9 | gRNA | AcrIIA4-mAID | auxin | AcrIIA4-mAID fusion is degraded by auxin, and cas9 becomes active. | Control of the dSpCas9-based gene expression was demonstrated in plant cells. | [100] | |
| Small-molecule enhancers of precise genome editors | |||||||
| Cas nuclease | RNA | Small molecule | Mode of action | Notes | Ref. | ||
| LbCas12a | crRNA | VE-822, AZD-7762 | ATR kinase inhibitor, CHEK1 kinase inhibitor | Enhanced the editing efficiency up to 6-fold in human pluripotent stem cells. Not cytotoxic. | [103] | ||
| AsCas12a | crRNA | Quinazoline-2,4(1H,3H)-dione] | The compound stabilizes the Cas12a-crRNA complex. | Enhanced the editing efficiency up to ~1.8 fold in human cells. | [104] | ||
| Base editor | gRNA | Ricolinostat, nexturastat A | HDAC inhibitor | Induces robust base editing in diverse cell types, including human primary T cells and mouse embryos. Increased the expression level of base editors. | [105] | ||
| Base editor | gRNA | Nexturastat A, abexinostat | HDAC inhibitor | Enhanced the base-editing efficiency. Enhanced the product purity of BE3 by suppressing C-to-G conversion. | [106] | ||
| Base editor | gRNA | Romidepsin | HDAC inhibitor | Enhanced the base-editing efficiency. Enhanced the product purity of BE3. | [107] | ||
| Prime editor | pegRNA | Nexturastat A, vorinostat, abexinostat | HDAC inhibitor | Enhance the prime editing for deletions and insertions, but not for point mutations. Genomic loci-dependent enhancemnt. | [106] | ||