Table 1.
Potential inducible CRISPR-Cas9 systems for somatic cell genome editing
| Inducer/small molecule | “Leakiness” (degree of uninduced editing) | Degree of editing upon inducer addition | Animal model(s)/cell type(s) used to test system | Advantages | Disadvantages | Reference |
|---|---|---|---|---|---|---|
| Cre-controlled CRISPR (3C) mutagenesis system | not reported | not reported | zebrafish embryos (RPE cells or in neural crest-derived melanocytes) | • heat treatment is also needed to display gene editing | • has not been tested in human cells | Hans et al.59 |
| • zebrafish line can be used for conditionally controlled tissue-specific biallelic gene inactivation | • cannot be used for somatic cell editing in humans due to their inability to produce cre-recombinase | |||||
| 2xTetO DOX-inducible sgRNA system | low (0%–14%) | 20%–80% (high cleavage activity) (activity score >0.5 for most cell types) | L-363, MC-38, A-498, LL/2, LP-1, 786-0, NCI-H1299, CT26, 4 TI, HEK293T, mouse (spleen cells, bone marrow cells, blood cells) | • can be used for high-throughput genetic screening | • variable editing efficiency in different cells | Sun et al.71 |
| • mouse model available for this method | ||||||
| • DOX can be paired with IPTG-inducible systems to enable modulation of two genes independently | ||||||
| Cre-recombinase doxycycline | not reported | not reported | mouse/mESCs | • can be light activated | • non-reversible | Dow et al.74 |
| • temporal advantage since system is regulated by doxycycline | • diffusion of small molecules in and out of cells causes off-target recombination | |||||
| • DOX at higher concentrations has been shown to cause cytotoxic effect in human cell lines | ||||||
| • cannot be used for somatic cell editing in humans due to their inability to produce cre-recombinase | ||||||
| Self-inactivating CRISPR (SiC) with doxycycline | not reported | on-target/off-target editing 0.8 to 1.3 after addition of doxycycline | myeloid and lymphoid cells in vivo, both in mouse peripheral blood and bone marrow as well as in vitro human lymphocytes (HL-60s) | • Cas9 on and off method exists | • system takes days to achieve max Cas9 activity, so there is a longer time for not only on-target editing but off-target cleavages as well | Kelkar et al.75 |
| ObLiGaRe doxycycline-inducible SpCas9 (OdInCas9) | no Cas9 background activity | not reported | hiPSC, HCT116, HEK293, HepG2, A549, OVCAR8, N2a cells | • inducible Cas9 expression | • off-target recombination | Lundin et al.77 |
| • reversibility with DOX withdrawal | • not able to use for somatic cell editing in humans due to Tet systems not being able to be reproduced in humans | |||||
| • low immune response with use of AAV to deliver sgRNA | ||||||
| Split FK506 binding protein 12/FKBP rapamycin binding (FKBP/FRB) system | 10% | fewer off-target indels (5%–10%) as compared to WTCas9 (27%) | HEK293FT | • inducible Cas9 expression | • auto-assembly problematic when half of Cas9 not nuclearized | Zetsche et al.72 |
| • rapamycin is crucial to provide temporary immunity against Cas9 | • non-covalent protein dimerization | |||||
| 4-Hydroxytamoxifen (4-OHT) with split FKBP/FRB system | none to low background activity | 25% Cas9 activity | HEK293T | • high tunability | • no reversibility | Nguyen et al.73 |
| • split systems can easily be delivered in multiple plasmids for efficient delivery | • Cas9 remains constitutively active | |||||
| • rapamycin could reduce the immune response against Cas9 | ||||||
| 4-OHT-responsive intein for Cas9 | low | 25-fold higher specificity (on-target/off-target indel frequency) | HEK293 cells | • improved specificity | • irreversible | Davis et al.81 |
| • split proteins can form aggregates due to exposed hydrophobic core | ||||||
| iCas system | low (but higher than split-Cas9 and comparable to intein-Cas9) | 38%–60% editing | HEK293 | • higher editing efficiency (as compared to split Cas9 and 4-OHT-responsive intein-mediated Cas9 at multiple loci) | • high background activity | Liu et al.82 |
| • reversible | ||||||
| Allosterically regulated Cas9 (arC9) | no background | 30% editing | E. coli, HEK293T, murine BNL CL.2 cells | • reversible | • slow reversibility (took 2 days) | Oakes et al.83 |
| Degradation tag (dTAG) system | low | on-target/off-target editing is enhanced (depending on the site, 1.5-fold to 4-fold as much compared to WT Cas9) | HEK293T, U2OS.eGFP.PEST, Drosophila S2 cells, mESC cell line | • small molecule activation provides temporal control | • while said to be reversible, there is no data on reversibility with Cas9 | Sreekanth et al.88 |
| • Cas9 target specificity is enhanced by addition of dTag | • tested in only a few organisms | |||||
| • degradation of Cas9 allows for control of editing outcome | ||||||
| • reversible | ||||||
| Small molecule-Controlled Cas9 repressible system | not reported | on-target/off-target editing is enhanced | HEK293T cells | • reversible | • existing Cas9 cannot be degraded | Wu et al.87 |
| • no structural modification from tagging | • exact mechanism is unknown | |||||
| • easy to measure half-life kinetics | ||||||
| Auxin-induced degron (AID) system | low background with auxin absent | not reported | HEK293FT, CHO-K1 | • makes tissue-specific activity controllable | • receptor (osTIR1) might be immunogenic | Kleinjan et al.68 |
| • reversibility with auxin removal when using miniAID system | • high dosages of auxin osTIR1 needed to attain degradation | Li et al.85 | ||||
| • minimal basal degradation | • IAA has been shown to cause toxicity at high amounts100 |
Selected genome editing studies that have been conducted using small molecule regulation are compared here. Leakiness, degree of editing upon inducer addition, and animal model/cell type used to test the system are summarized, with the reference indicated on the right. Leakiness is the degree of editing that occurs when the small molecule is not added to activate the system. Animal/cell models: RPE, retinal pigment epithelium; L-363; human plasma cell leukemia cell line; MC-38, C57BL/6 murine colon adenocarcinoma cell line; A-498, Homo sapiens kidney carcinoma cell line; LL/2, murine Lewis lung carcinoma cell line; LP-1, human myeloma cell line; 786-0, kidney adenocarcinoma cell line; NCI-H1299, human non-small cell lung carcinoma cell line; CT26, undifferentiated colon carcinoma cell line; 4T1, murine breast cancer cell line; HEK293T/FT, human embryonic kidney cell line; HL-60, human lymphocyte line; hiPSC, human-induced pluripotent stem cell; HCT116, human colon cancer cell line; HepG2, human liver cancer cell line; A549, adenocarcinoma human alveolar basal epithelial cell line; OVCAR8, human ovarian carcinoma cell line; N2a, mouse neuroblastoma cell line; U2OS, Homo sapiens bone osteosarcoma; CHO-K1, Chinese hamster ovary cell line.