Strategies for site-specific recombination with high efficiency and precise spatiotemporal resolution

Abstract

Site-specific recombinases (SSRs) are invaluable genome engineering tools that have enormously boosted our understanding of gene functions and cell lineage relationships in developmental biology, stem cell biology, regenerative medicine, and multiple diseases. However, the ever-increasing complexity of biomedical research requires the development of novel site-specific genetic recombination technologies that can manipulate genomic DNA with high efficiency and fine spatiotemporal control. Here, we review the latest innovative strategies of the commonly used Cre-loxP recombination system and its combinatorial strategies with other site-specific recombinase systems. We also highlight recent progress with a focus on the new generation of chemical- and light-inducible genetic systems and discuss the merits and limitations of each new and established system. Finally, we provide the future perspectives of combining various recombination systems or improving well-established site-specific genetic tools to achieve more efficient and precise spatiotemporal genetic manipulation.

Keywords: Cre-loxP; Dre-rox; gene manipulation; genome engineering; inducible; lineage tracing; optogenetics; photoactivatable; site-specific recombinase; site-specific recombination.

Figure 1

Schematic of the Cre-loxP recombination mechanism.A, the loxP site is a 34 bp consensus sequence and consists of a central 8 bp nonsymmetrical spacer flanked by two 13 bp palindromic recognition sites. Cre recombinase subunits bind each palindromic sequence and cleavage, exchange, and ligation DNA strand at the central spacer. The spacer provides the orientation of the loxP site. The nucleophilic tyrosine 324 in Cre recombinase attacks the phosphate forming a three phosphotyrosine bond and releasing a free 5′ OH. Cleavage position is indicated by arrow. B, a dimer of Cre subunits bind at the palindromic sequence of each loxP site. A tetrameric Cre structure arranges two loxP sites in an antiparallel fashion to stabilize a synaptic complex. Two opposite Cre subunits cleave and exchange one pair of strands. The released 5′ OH attacks the neighboring strand to form a Holliday junction intermediate. The second pair of Cre subunits is activated by an isomerization of the Holliday junction intermediate. The second pair of strands are cleaved and exchanged to complete the recombination and result in the recombinant products. C, different outcomes of a Cre-loxP recombination depending on the position and orientation of the two loxP sites. If the two loxP sites are in the same orientation, the recombination results in the excision or integration of the DNA segment (e.g., target gene) flanked by the two loxP sequences. If the orientation of loxP elements is in opposite, the result of the reaction is the inversion. Recombination between the two loxP sites located on different DNA strands results in the exchange or translocation.

Figure 2

Chemical-inducible recombination systems.A, a self-cleaved inducible CreER (sCreER) with a Cre-loxP-ER-loxP construct that switches inducible CreER into a constitutively active Cre by itself once tamoxifen induction. B, the iSuRe-Cre is an inducible dual reporter-Cre, containing CAG promoter, N-PhiM and MbTomato reporter gene, and a constitutively active and permanently expressed Cre. After removal of the floxed N-PhiM cassette by Cre or CreER, iSuRe-Cre co-expresses MbTomato and a constitutively active Cre and significantly increases the efficiency and certainty of gene deletion in reporter-expressing cells. C, the DiCre in which Cre is split into two inactive moieties and fused with FKBP12 and FRB respectively. FKBP12 and FRB can be heterodimerized efficiently after rapamycin treatment, leading to the complementation of inactive fragments (CreN and CreC) and Cre activity restoration. D, the Roxed-Cre contains a Cre-N-rox-stop-rox-Cre-C construct. The reinstatement of Cre activity occurs after the removal of the rox-flanked STOP cassette by Dre-rox recombination. E, the CrexER with a Cre-rox-ER-rox construct executes Cre activity after the removal of ER by Dre-rox recombination. F, the Tet-On inducible system consists of two transgenes, a recombinase under the control of a TRE and a rtTA driven by a cell-specific promoter. After Dox administration, the rtTA is active and interacts with the TetO promoter. CreC, the C-terminal domain of Cre; CreN, the N-terminal domain of Cre; DiCre, dimerizable Cre; Dox, doxycycline; FKBP12, FK506-binding protein; FRB, binding domain of the FKBP12-rapamycin associated protein; rtTA, reverse tet-controlled transactivator; Tam, tamoxifen; TRE, Tet responsive element.

Figure 3

Enhanced efficiency of gene knockout by sCreER, compared with conventional CreER.A, schematic figure showing experimental design. B, schematic diagram showing Kdr gene deletion by sCreER and CreER. C and D, immunostaining for tdTomato, VEGFR2, and CDH5 on heart sections from E15.5 Npr3-sCreER;Kdr^flox/flox; R26-tdTomato and Npr3-CreER;Kdr^flox/flox; R26-tdTomato mouse embryos. Quantification data showed the percentage of VEGFR2⁺ endocardial cells in CDH5⁺ endothelial cells in the inner myocardial wall. ^∗p < 0.05; Data are mean ± SEM; n = 5. Scale bar, 50 μm. Each image is a representative of five individual mouse samples. endo, endocardium; Ex, exon; LV, left venticle; sCreER, self-cleaved inducible CreER; Tam, tamoxifen.

Figure 4

Genetically encoded light-inducible recombination systems.A, CRY2 and the N terminus of CIB1 are fused to the CreN and CreC, respectively. Upon the illumination of blue light, the dimerization of CRY2 and CIBN leads to the reconstitution of split Cre recombinase activity. B, the photoreceptor Vivid (VVD) is designated as Magnets and comprises two photoswitches named pMag and nMag. The heterodimerization of pMag and nMag induced by the blue light illumination leads to the complementation of CreN and CreC and Cre activity reconstitution. C, the PhyB absorbs red and infrared light through the photoisomerization of a covalently bound PCB. The conformation of PhyB changes between the Pr (red-absorbing) and Pfr (far-red-absorbing) states catalyzed by red and infrared light. The PIF only associates with PhyB in Pfr state. The heterodimerization between PhyB and PIF is reversibly triggered by red (650 nm) and infrared (750 nm) light. D, a light-inducible transcription activation system based on BphP1-PpsR2 and TetRtetO. BphP1-mCherry and the C terminus of NLS-PpsR2 are fused to the TetR and VP16, respectively. Upon near-infrared light illumination, BphP1 converts into the Pr state and forms heterodimer with PpsR2. NLS facilitates the heterodimer translocates to the nucleus where BphP1 fusions interact with tetO DNA repeats via TetR. VP16 recruits the transcription initiation complex and triggers gene transcription. E, the FISC system is designed on the basis of the affinity of bacteriophytochrome Coh2 and DocS. In this system, DocS-CreC fusion protein is under the control of FRL-inducible promoter PFRL, CreN fused to Coh2 driven by a constitutive promoter PhCMV. Upon FRL exposure, the active photoreceptor BphS converts GTP into c-di-GMP which induces binding of FRTA (p65-VP64-BldD) to promoter PFRL to drive DocS-CreC expression. The interaction of Coh2 and DocS domains leads to the reunion of CreC and CreN and Cre activity reinstatement. c-di-GMP, cyclic diguanylate monophosphate; CreC, the C-terminal domain of Cre; CreN, the N-terminal domain of Cre; CRY2, cryptochrome 2; FISC system, far-red light-induced split Cre-loxP system; FRL, far-red light; FRTA, far-red light-dependent transactivator; GTP, guanylate triphosphate; NLS, nuclear localization signal; nMag, negative Magnet; PCB, chromophore phycocyanobilin; PhyB, photoreceptor phytochrome B; PIF, phytochrome interaction factor; pMag, positive Magnet; TetR, tetracycline repressor.

Figure 5

Decision tree for choosing an appropriate site-specific recombination system or strategy. Flow chart summarizing site-specific recombination strategies discussed in the text and Table 1. CrexER, a switchable CreER system with the Cre-rox-ER-rox construct; Di-Cre, dimerizable Cre; iSuRe-Cre, a Cre/CreERT2-inducible dual Reporter-Cre-expressing mouse allele; 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.