One technique that is often heard or used in the manipulation of molecular expression is:Cre LOXP or FLP FRT recombinase system。In simple terms, this type of recombinase system mediates the recombination between recombination tar-get sites (RTS) through site-specific recombinases (SSRS) to achieve gene knockout, gene insertion, gene transversion, and gene translocation at specific sites. Because this technology can effectively overcome the shortcomings of other types of recombinant technologies, such as non-specificity or low recombination efficiency, it has gradually occupied a dominant position in the field of functional gene research in recent years.
The basic principle of cre-loxp
CRE (Cyclization Recombination Enzyme) is a recombinase, ** in P1 bacteriophage, its gene coding region sequence is 1029BP long, 38 kDa size, polypeptide monomer protein composed of 343 amino acids. The C-terminal domain of CRE recombinase contains catalytic active sites, which can catalyze recombination between specific sites in the DNA molecule, and at the same time, CRE can also recognize specific DNA sequences, i.e., LOXP sites, so that gene recombination can occur between two LOXP sites.
LOXP is the abbreviation of Locus of X-Overp1, which is a 34 bp long sequence located in the P1 phage, which is composed of two 13 bp reverse palindromic sequences and 8 bp intermediate spacer sequences, the reverse palindromic sequence is the recognition and binding region of the CRE recombinase, and the spacer sequence determines the direction of the LoxP sequence.
Ways in which CRE LOXP induces genetic recombination
In general, when there are two LOXP sites in the cell genome, CRE recombinase induces sequence recombination between the two LOXP sites. The result of the recombination depends on the orientation of the two loxp sites, and there are several main possibilities:
If the two LOXP sites are on the same DNA strand and are oriented in the same direction, the CRE recombinase can effectively delete the deletion between the two LOXP sites (Figure 1A).
If two LOXP sites are on the same DNA strand but facing opposite directions, CRE recombinase can induce sequence inversion between the two LOXP sites (Figure 1B).
If two LOXP loci are located on two different DNA strands or chromosomes, CRE recombinase can induce the exchange or chromosomal translocation of the two DNA strands, i.e., gene translocation (Figure 1C).
If the four LOXP sites are located on two different DNA strands or chromosomes, CRE recombinase can induce cassette exchange between LOXPs (Figure 1D).
Fig.1 Ways in which cre-loxp induces gene recombination.
The Cre-LOXP system can realize the manipulation of genes, and according to the way in which the CRE recombination system induces gene recombination, we can achieve CRE-dependent gene expression through the following three strategies:
1. LSL sequence (conditional gene selection).
LOXP2 and transcription stop cassette were inserted between the promoter and the gene of interest, and there was a co-directional LOXP site at each end of the transcription stop cassette to form the LOXP-stop-LOXP-gene pattern, i.e., the LSL sequence.
In this case, in cells without the presence of CREase, the target gene downstream of the transcription termination signal box is not expressed at all, but if the cell contains CREase, the deletion process in gene recombination occurs, and the transcription termination signal box is removed, and the target gene is expressed. This is a simple and effective way to selectively express genes in certain cells through the LSL setting.
Fig.2 Cre-dependent gene expression-LSL strategy.
2. dio do sequence
By introducing two pairs of incompatible incompatible inverted lox sites, loxp and lox2272, a steady state can be reached after two rounds of recombination of the two sets of lox sites. That is, the expression of genes can be controlled by the presence or absence of CRE recombinase.
This strategy is known as dio (cre-on) or do (cre-off). Under this strategy, the sequence between any pair of LOX sites will be reversibly and rapidly flipped under the action of CRE, and then the CRE recombinase will immediately and irreversibly cleave the flipped co-rotating LOX site, leaving only the flipped gene and the separate LOXP and LOX2272 sites to prevent the gene from being flipped again. This ingenious design allows researchers to construct dio and reverse genes in viral vectors, and after infecting cre-positive cells, cre-positive cells can be allowed to express genes, which are dio structuresIf the prepackaged gene is positive, then the cre-positive cells do not express the gene, and the rest of the cells express the gene, which is the DO structure. As a result, manipulating gene expression has become a reality.
Fig.3 Cre-dependent gene expression was achieved with the flex strategy of LOXP and LOX2272.
scott m. sternson, et al., j. neurosci., 2008）
3. MADM system
The MOSAIC Analysis with the Double Markers (MADM) system is based on the process of CRE-induced translocation. By homologous recombination, two chimeric marker genes (fluorescent proteins) are mapped to the same site on the homologous chromosome, and a LOXP site is inserted between the N-terminus (C-terminus) of a fluorescent protein and the N-terminus (C-terminus) of another fluorescent protein on the same DNA strand. In the absence of CRE, functional GFP or RFP are not expressed;However, when Cre is expressed in specific cells at a specific time, CRE can induce DNA strands with two different fluorescent protein N-terminus and another DNA strand with the same fluorescent protein C-terminus in cells** Recombination occurs in the G2 phase so that two daughter cells each express either a functional fluorescent protein (called x-segregation), or one of the daughter cells expresses two fluorescent proteins and the other daughter cell does not have any functional fluorescent protein (term: z-segregation). In addition, recombination can occur in the G1 or G0 phases of the cell cycle, in which case the cells express both fluorescent proteins simultaneously.
Figure 4 MADM system.
zong hui, et al., cell., 2005）
Application of the CRE LOXP system
The introduction of a CRE or LOXP element into a virus in combination with a transgenic mouse can be used to specifically label or genetically manipulate a certain type of cell.
LSL policy applies
The MT Mg (ROSA-PCA-LOXP-MT-PA-LOXP-MG-PA) rat is a dual-fluorescent reporter mouse that normally expresses a red fluorescent protein (TDTOMATO) localized to the membrane and a green fluorescent protein (GFP) localized to the membrane when the cells express CRE.
ALB is a specific promoter of adult animal hepatocytes, and mice injected with the tail vein of Ra**2 8-ALB-Cre virus MT MG mice can be found to be specifically ** infected in mouse liver tissue (green), while heart tissue is not infected (shown in red).
Fig.5 Liver tissue (left) and heart tissue (right).
Professor Guan Jisong's team injected A**-SYN-CRE into the right AUD region of ASH1LFL FL mice to conditionally knock out the ASH1L gene, and found that ASH1L mediates the activity-dependent synaptic pruning process of cortical neurons in a cellular autonomous effect. （Click to read: neuron |Save the Children of the Stars!The Jisong group of the University of Science and Technology revealed the neural mechanism of autism caused by ASH1L haploinsufficiency in mice
Fig.6 A**-Cre injected with flox mice to achieve conditional knockout.
yan yuze, et al., neuron., 2022）
DIO policy application
Thy1 is a specific promoter of pyramidal neurons, which is abundantly expressed in the forebrain, hippocampus, amygdala, thalamus and retina.
Fig.7 A**-dio injected into thy1-cre mice to achieve specific labeling.
When using neural tracing technology to elucidate the structure and function of specific neural circuits in the brain, the CRE recombinase system can also be used in combination with A** serotypes (such as Ra**2 9, Ra**2 Retro, Ra**2 1) to achieve the purpose of specific neural circuit markers and functional studies.
Fig.8 The cre-dio system combined with virus tracing technology to achieve the labeling and regulation of specific loops.
ma hui, et al., proc natl acad sci u s a., 2021）
MADM system applicationWith the help of the cardiomyocyte-specific promoter TNNT2 to drive the expression of CREase, the state of cardiomyocyte cells** was tracked with MADM-ML-11TG GT mice. （Click to read: [Cell Stem Cell] Cocktail** promotes cardiomyocyte proliferation and heart regeneration
Figure 9 Application of the MADM system.
du jianyong, et al., cell stem cell., 2022）
List of CRE viruses (partial).
Retrofit Cre LoXP system
After the modification of CRE and LOXP elements by modern genetic engineering methods, the CRE LOXP system has achieved a richer conditional recombination strategy.
1. Transformation of CRE components:The modification of the CRE element improves the activity of CRE recombinase and achieves drug inductibility. For example, by introducing the eukaryotic nuclear localization sequence NLS on the CRE element, CRE enables recombination at low expression abundances, which is important for some low-abundance promoters. In addition, after the C termination of the CRE element is connected to a modified ligand-binding domain LBD, the new fusion protein Cre-LBD will be localized in the cytoplasm, when the synthetic hormone molecule binds to the Cre-LBD receptor, the protein conformation changes and enters the nucleus, mediating gene recombination, the most commonly used is the Creert2 mutant induced by Tamoxifen, its LBD comes from the estrogen receptor ER molecule, when there is Tamoxifen, CRE can mediate gene recombination, so as to control the injection time of Tamoxifen, which can realize the time-specific regulation of gene recombination.
Figure 10 Tamoxifen-induced Cre-ER system.
hyeonhui kim, et al., lab anim res., 2018）
2. Far infrared light-induced split cre-loxp system:Chemically induced Cre-LOXP has disadvantages such as cytotoxicity, leakage, and off-target, and the team of researcher Haifeng Ye of East China Normal University established a far-red light-induced split cre-loxp system (FISC) based on the split-cre recombinant system and far-infrared light-inducible optogenetic system. In this system, the Cre enzyme is divided into two fragments, and the N-terminal Cre is fused to a CoH2 structure (CreN-CoH2);C-terminal CRE fuses into a DOCS-CREC;When FRL is illuminated, COH2 and DOCs bind under strong affinity, and the CREzyme is also recombined to perform functions. This system achieves the purpose of conditional non-invasive gene regulation. （Click to read: [nat commun.]】Innovative research of Ye Haifeng's team at ECNU: non-invasive far-infrared light-induced split-cre system regulates gene recombination
Fig.11 FISC system.
wu jiali, et al., nat commun., 2020）
3. Transformation of LOXP componentsThere are also some mutants of the LOXP element, the spacer and the palindromic sequence can be mutated, and the mutated sequence can still be recognized and recombined by CRE, but the mutated LOXP sequence must match the same mutated LOXP sequence to mediate gene recombination, and cannot match the unmutated LOXP sequence, so that the combination of different LOXP sequences can be used to control multiple genes (such as the LOX2272 site described above), and under the action of the same CRE recombinase, multiple sequences of gene recombination can be realized, resulting in very diverse recombination results。 For example, the brilliant and colorful fluorescent labeling effect of brainbow technology is officially based on the modification of the LOXP sequence.
Other recombinant enzyme systems
In addition to the Cre-Loxp system, there are similar VCRE-VLOXP and SCRE-SLOXP systems, as well as the FLP-FRT F5 system and the DRE-ROX1 Rox2 system, which have no cross-influence with the Cre-Loxp system, and the corresponding expression schemes are FDIO and DDIO systems, respectively. The existence of multiple recombinase systems provides convenience for the design of multiple constraints in the study, and the flexible application of Cre, FLP, and DRE recombinase systems will help to carry out more in-depth topics (To learn more about recombinant enzymes, click to read: [Knowledge Sharing] Half seawater, half flame, molecular expression controls how recombinant enzyme systems such as CRE, FLP, DRE and other recombinant enzyme systems that appear at medium and high frequencies work
Fig.12 Comparison of CRE FLP DRE systems.
lief e fenno, et al., nat methods., 2014）
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