Table 1.
An assessment of currently used and emerging recombination system
| Recombination systems | Requirements | Merits | Limitations | Improved production | Refs | |
|---|---|---|---|---|---|---|
| Chemical-inducible system | ||||||
| sCreER | Tamoxifen induction | High efficiency; temporal control; reduced toxicity of tamoxifen | The efficiency of the initial CreER-loxP recombination-mediated switch of sCreER into Cre depends on the recombination efficiency of the loci where the sCreER knock-in. | Efficient and temporally controlled gene deletion for functional study. | (2) | |
| iSuRe-Cre | Cross w/other Cre/CreER lines; w/or w/o tamoxifen induction | High efficiency; temporal control; nontoxicity; no leakiness; reliably reports cells with gene deletion; compatible with the numerous existent loxP and Cre/CreERT2 alleles | Does not prevent the occurrence of gene-deletion false-negatives | Increasing the efficiency and reliability of Cre-dependent reporter and gene function analysis. | (3) | |
| Di-Cre | Rapamycin induction | Tight temporal control; rapid induction; low background Cre activity | Toxic during development; only can be used in adult animals | Tight temporal control of recombinase activity for conditional gene knock-out. | (47, 48) | |
| Roxed-Cre | Dual recombinases, Split-Cre and Dre | High-resolution recombination; ideal for sequential lineage tracing | A rox site remained in the coding sequence of Cre may decrease Cre activity; cannot be controlled temporally | The binary SSR system is ideal for sequential lineage tracing studies aimed at unraveling the relationships between cellular precursors and mature cell types. | (24) | |
| CrexER | Dual recombinases, Cre and Dre | Organ- or tissue-specific gene manipulation | Cannot be controlled temporally | The intersectional genetic system achieves both gene knockout and overexpression in vascular endothelial cells in an organ- or tissue-specific manner. | (25) | |
| Tet-On system | Genetically modified lines; Dox induction | Less toxic | Complexity of mice crossing; time-consuming | Temporal, spatial, and cell type-specific control of gene expression. | (49, 50, 51) | |
| Light-inducible system | ||||||
| System based on caged biomolecules | ||||||
| Photocaged 4-OHT or analogs | UV light illumination | Precise spatiotemporal control of gene expression | Cytotoxicity (DNA damage) by UV light; poor tissue penetration; limited recombination efficiency | Precise temporal- and location-specific control of CreER-mediated recombination in a light-dependent manner. | (61, 62, 63) | |
| Caged doxycycline/cyanodoxycycline | UV light or two-photon illumination | Precise spatial and temporal control of gene expression; the amount of UV light needed for induction is innocuous | Diffuse background fluorescence; low membrane permeability | High-resolution conditional transgene expression ranging from single cells to entire organisms. | (66) | |
| Near-IR uncaging strategy based on cyanine photochemistry | Near-IR light illumination | Cytocompatible; tissue penetration | Need to be validated in vivo | Spatial and temporal control of drug delivery. | (67) | |
| Light-cleavable dRap | UV light illumination | Simple to set up; temporal control of gene expression | Could not be readily placed under photochemical control | Protein dimerization induced by optically activated rapamycin dimer can be applied to control recombinase function. | (68) | |
| Photocaged Cre recombinase | Non-photodamaging UVA light illumination | Spatiotemporal control; background-free | Need to be validated in vivo | Tight spatiotemporal control of the activity of Cre recombinase and DNA recombination. | (70, 71) | |
| Genetically encoded system | ||||||
| CRY2-CIB1 system | PA-Cre 1.0 | Blue light illumination | Fast temporal; subcellular spatial resolution; reversible | Inefficient packaging; poor penetrative capacity | Fast temporal and spatial resolution without the need for exogenous cofactors. | (74) |
| PA-Cre 2.0 | Blue light illumination | High induced activity; low background; a single and brief light pulse; reduced light-mediated toxicity; reversible | Low recombination efficiency; need tuning nuclear import/export signals to reduce sensitivity to expression level differences to attain low background | Five-fold improved activity allowing precise spatial and temporal control of Cre recombinase ranging from single cells to whole organisms. | (92, 93) | |
| Li-rtTA | Dual induction of blue light and Dox | Reversible; spatiotemporal specific | Complexity of mice crossing; time-consuming | Genetical labeling and lineage tracing of multiple cell types in regional skin in a spatiotemporally specific manner. | (95) | |
| VVD system | LightOn | Blue light illumination | Simple and robust; spatiotemporal control; reversible | Poor penetrative capacity | A simple and robust system to quantitatively and spatiotemporally control gene expression and manipulate many biological processes in living cells and organisms. | (96) |
| Magnet | Blue light illumination | Low dimerization activity in the dark state; high spatiotemporal precision; reversible | Poor penetrative capacity | Spatially and temporally precise control over several signaling proteins in living mammalian cells with substantially enhanced dimerization efficiencies and accelerated switch-off kinetics. | (78) | |
| PA-Cre 3.0 | Blue light illumination | Reduced dark leak activity; improved efficiency; reversible | Poor penetrative capacity | Significantly addressed the issues of low recombination efficiency and dark leakiness. | (94) | |
| PhyB-PIF system | Red/far-red light illumination | Rapid stimulation and reversibility | Need for exogenous cofactor | Precisely and reversibly control gene expression and cell signaling. | (73, 100) | |
| BphP1-PpsR2 system | Near-IR light activation | Deep tissue penetration; low phototoxicity; tetracycline-independent | Minor interference with cellular metabolism | Targeting subcellular protein, inducing intracellular enzymatic activity, and activating gene expression with deep tissue penetration and low phototoxicity. | (101) | |
| FISC system | Far-red light excitation | High recombination efficiency; spatiotemporal precision; low background and photocytotoxicity; deep penetration capacity | Complexity; may require developing a vector with expanded packing capacity and small construct size to ensure efficient delivery in vivo | Precise control of genome engineering in target single cells or whole organisms in a spatiotemporal fashion with deep penetration, reduced toxicity, and invasiveness. | (102) | |
4-OHT, 4-hydroxytamoxifen; BphP1, bacterial phytochrome; CIB1, a basic helix-loop-helix protein; CrexER, a switchable CreER system with the Cre-rox-ER-rox construct; CRY2, cryptochrome 2; Di-Cre, dimerizable Cre; Dox, doxycycline; dRap, light-cleavable rapamycin dimer; FISC system, far-red light-induced split Cre-loxP system; iSuRe-Cre, a Cre/CreERT2-inducible dual Reporter-Cre-expressing mouse allele; Li-rtTA, light activated rtTA; LightOn, the light-on system; Near-IR, near-infrared; PA-Cre, photoactivatable Cre recombinase; PhyB, photoreceptor phytochrome B; PIF, photochrome-interacting factor; PpsR2, the natural partner of BphP1; Roxed-Cre, a sequential binary SSR system with the Cre-N-rox-stop-rox-Cre-C strategy; sCreER, self-cleaved inducible CreER with a Cre-loxP-ER-loxP construst; Tet-On system, tetracycline-inducible gene expression system; VVD, photoreceptor Vivid.