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How Does Cre, Flp, Dre Recombinase System Work?

With the deepening of neuroscience research, scientists need to pay attention to many different types of neurons or glial cells, such as a variety of GABAergic interneurons and microglia. Unfortunately, these cells often do not have mature, non-leaking promoters to choose from. Therefore, it is very important to achieve cell type-specific labeling or manipulation. Recombinase systems such as Cre-LoxP or Flp-FRT are ideal tools for labeling specific cell types.

Basic Principle of Cre-loxP

Cre (Cyclization Recombination Enzyme) is a recombination enzyme that was discovered from P1 phage in 1981. Its gene coding region has a total length of 1029 bp and is a 38kDa polypeptide monomer protein composed of 343 amino acids. The C-terminal domain of Cre recombinase contains catalytically active sites, which can catalyze recombination between specific sites in DNA molecules.

LoxP is the abbreviation of Locus of X-overP1. It is a 34bp sequence located in the P1 phage, which is composed of two 13bp reverse palindrome sequences and an 8bp intermediate spacer sequence. The reverse palindrome sequence is the recognition and binding region of Cre recombinase, and the spacer sequence determines the direction of the LoxP sequence.

To put it simply, this type of recombinase system uses site-specific recombinases (SSRs) to mediate recombination between Recombination tar-get sites (RTs) to achieve specific sites manipulation, such as gene knockout, gene insertion, gene flip, and gene translocation. Since this technology can effectively overcome the shortcomings of non-specificity or low recombination efficiency, it has gradually occupied a dominant position in the field of functional gene research in recent years.

Cre recombinase not only has catalytic activity but is similar to restriction enzymes. It can recognize specific DNA sequences, namely loxP sites so that gene recombination occurs between two loxP sites. Cre recombinase does not require any cofactors, and can act on DNA substrates of various structures, such as linear, circular, and even supercoiled DNA. In addition, the activity of Cre recombinase is controllable, so that it can be expressed only in a certain type of cell type, or it can be triggered by specific external stimuli (such as chemical signals, thermal stimuli). In the field of neuroscience, this technology can be used for specific labeling of neural circuits, functional research of endogenous genes in specific cell types, and construction of mouse models.

Several Ways of Cre-loxP Inducing Gene Recombination

Generally, when there are two LoxP sites in the cell genome, Cre recombinase will induce sequence recombination between the two LoxP sites. First, Cre recombinase binds to two 13bp palindrome sequences to form a dimer, and then this dimer combines with another dimer at the loxP site to form a tetramer. The LoxP site is oriented, and the two loxP sites forming the tetramer are parallel. Then the double-stranded DNA between the two loxP sites is cut off by Cre recombinase, and the nick is under the action of DNA ligase. reconnect. The result of recombination depends on the orientation of the two loxP sites, and there are mainly the following possibilities:

LoxP is the abbreviation of Locus of X-overP1. It is a 34bp sequence located in the P1 phage, which is composed of two 13bp reverse palindrome sequences and an 8bp intermediate spacer sequence. The reverse palindrome sequence is the recognition and binding region of Cre recombinase, and the spacer sequence determines the direction of the LoxP sequence.

① If two LoxP sites are located on a DNA strand and the directions are the same, Cre recombinase can effectively delete the sequence between the two LoxP sites (Deletion) (Figure 1A).
② If two LoxP sites are located on a DNA strand but in opposite directions, Cre recombinase can induce sequence inversion between the two LoxP sites (Figure 1B).
③If the two LoxP sites are located on two different DNA strands or chromosomes, Cre recombinase can induce the two DNA strands to exchange or chromosomal translocation, that is, gene translocation (Figure 1C)
④ If the four loxP sites are located on two different DNA strands or chromosomes, Cre recombinase can induce cassette exchange between loxP (Figure 1D).

The way Cre-loxP induces gene recombination Figure 1 The way Cre-loxP induces gene recombination.

According to the way the Cre recombination system induces gene recombination, Cre-dependent gene expression can be achieved through the following two strategies:

  1. LSL sequence
  2. Insert LoxP2 and a transcription stop cassette (Transcription STOP cassette) between the promoter and the target gene. There is a LoxP site in the same direction at both ends of the transcription stop cassette, forming the LoxP-STOP-LoxP-gene pattern, that is, the LSL sequence.

    In this case, in cells without Cre enzyme, the downstream target gene of the transcription termination signal box is not expressed at all, but if the cell contains Cre enzyme, the deletion process in gene recombination occurs, remove the transcription termination signal box, and then express the purpose gene. This is a simple and effective method. We can selectively (selected according to Cre strains) express genes in certain cells through the setting of LSL. However, the disadvantage of this method is that transcription leakage may occur, that is, the STOP sequence is skipped during transcription, and genes can be expressed without the action of Cre enzyme.

    Cre-dependent gene expression - LSL-based strategy Figure 2 Cre-dependent gene expression - LSL-based strategy

    Application of Cre-loxp System

    1. Specific Labeling/Neural Circuit Tracing
    2. It can be used in combination with Cre strain mice and Cre-dependent expression viral vectors to achieve specific labeling and manipulation of certain cells. When using neural tracing technology to analyze the structure and function of specific neural circuits in the brain, the Cre recombinase system can also be used in combination with AAV serotypes (such as rAAV2/9, rAAV2/retro, rAAV2/1) to achieve specific neural circuit labeling and functional research.

      Anterograde transsynaptic tracing based on rAAV1-Cre Figure 4 Anterograde transsynaptic tracing based on rAAV1-Cre (Zheng, 2019)

    3. Virus-dependent Genetic Recombination
    4. Due to the disadvantages of genetic recombination that transgenic animals rely on, such as time-consuming, high cost, and low tissue specificity, more and more scientific researchers are now beginning to use the Cre-loxP system in a more flexible way. The introduction of Cre or loxP elements through viruses, combined with a transgenic mouse is a more common way (Figure 5). Compared with gene recombination that transgenic animals rely on, virus-dependent gene recombination has the following advantages:

      Stronger tissue specificity: Since the virus can be injected locally to ensure region-specific infection, coupled with the specific promoter driving the Cre gene, it can achieve stronger region and cell-specific gene recombination.

      Less cost: The cost of buying genetically modified animals is generally more expensive, and the breeding of genetically modified animals and genotype identification require a lot of manpower and material resources, while the cost of virus preparation, storage and injection is relatively low.

      Shorter experimental period: Because the virus can act very quickly, and the mating process of transgenic mice is particularly time-consuming, the method of injecting the virus into the transgenic mice can greatly shorten the experimental period.

    LoxP animal combined with Cre expressing virus for gene knockout Figure 5 LoxP animal combined with Cre expressing virus for gene knockout.

    Advantages of Cre-loxP System

    The Cre-loxP system is the most widely used conditional gene knockout tool in the nervous system, mainly due to the following advantages:

    High efficiency: After Cre recombinase forms a complex with DNA fragments with loxP sites, it can provide enough ability to initiate the subsequent DNA recombination process. The recombination process is simple and efficient.
    High specificity: The loxP site is a 34bp element with a palindrome sequence structure and a gap in the middle. This structure ensures the uniqueness of the loxP sequence, thereby ensuring the strong specificity of gene recombination.
    Wide range of applications: Cre recombinase is a relatively stable protein that can play a role in different tissues and different physiological conditions in organisms.
    Can be expressed by type 2 promoter: Cre recombinase encoding gene can be driven by any type 2 promoter, thereby ensuring that Cre recombinase can be used in different cells, tissues and organs of the organism, thereby achieving high tissue and cell specificity.

    Modified Cre-loxP System

    Modified Cre-loxP System

    By using modern genetic engineering methods to transform Cre and loxP elements, the Cre-loxP system can achieve more abundant conditional recombination strategies.

    1. Modification of Cre element: The modification of Cre element improves the activity of Cre recombinase and realizes drug inducibility. For example, by introducing the eukaryotic nuclear localization sequence NLS on the Cre element, Cre recombinase can achieve recombination at low expression abundance, which is important for some low-abundance promoters. In addition, a modified ligand binding domain LBD is connected to the C-terminus of the Cre element. The new fusion protein Cre-LBD will be located in the cytoplasm. When the synthetic hormone molecule binds to the Cre-LBD receptor, The protein conformation changes and enters the nucleus to mediate gene recombination. At present, the CreERT2 mutant induced by tamoxifen is the most used. Its LBD comes from the estrogen receptor ER molecule. When tamoxifen is present, Cre can mediate gene recombination. In this way, by controlling the injection time of tamoxifen, the specific regulation of gene recombination time can be achieved.
    2. Cre-ER system induced by tamoxifen Figure 6 Cre-ER system induced by tamoxifen (Hyeonhui, 2018)

    3. Modification of loxP element: loxP elements also have some mutants. Both the spacer and palindrome sequences can be mutated, and the mutated sequence can still be recognized and recombined by Cre recombinase. Different loxP sequence combinations are used to control multiple genes (such as the lox2272 locus described above). Under the action of the same Cre recombinase, multi-sequence gene recombination can be achieved, resulting in very diverse recombination results. For example, the fluorescent labeling effect of Brainbow technology is based on the modification of the loxP sequence.

    Other Recombinase Systems

    In addition to the Cre-LoxP system, there are similar vCre-vLoxP, sCre-sLoxP systems, and the Flp-FRT/F5 system and Dre-Rox1/Rox2 system that have no cross-effects with the Cre-LoxP system. The corresponding expression schemes are respectively fDIO and dDIO systems. The existence of multiple sets of recombinase systems facilitates the design of multiple constraints in the research, and the flexible application of Cre, Flp, and Dre recombinase systems will facilitate the development of more in-depth topics (Figure 7).

    1. Flp-FRT system
    2. Flp-FRT system is similar to Cre-loxP in that it is also composed of a recombinase and a special DNA sequence. Among them, Flp (flippase recombination enzyme) recombination enzyme was found in yeast cells. The gene length is 1272bp with a 48kDa polypeptide monomer protein which composed of 423 amino acids.

      FRT is the recognition site of Flp, with a total length of 48 bp, consisting of three 13 bp reverse palindromes and an 8 bp spacer sequence (Figure 7a). The two reverse palindromes adjacent to the spacer sequence are the recognition and binding regions of Flp recombinase. The spacer sequence is the region where recombination occurs and also determines the direction of the entire sequence.

      The Flp-FRT system and Cre-loxP induce gene recombination in a similar way. The obvious difference between the two systems is that the recombinase (Cre and Flp) has different optimal reaction temperatures. Studies have found that Cre recombinase is the best The temperature is 37 oC and Flp recombinase is 30℃.

    3. Dre-Rox system
    4. Dre (D6 site-specific DNA recombinase) is also a tyrosine recombinase found in bacteriophages. It recognizes the 32bp DNA sequence rox, which contains two 14bp reverse palindromes and a 4bp intermediate spacer sequence (Figure 7a). Dre recombinase and Cre recombinase are specific, Dre recombinase cannot recognize loxp sites, and Cre recombinase cannot recognize rox sites.

      Comparison of Cre\Flp\Dre system. Figure 7 Comparison of Cre\Flp\Dre system. (Lief E Fenno, 2014)

    5. vCre-vloxp and sCre-sloxp system
    6. Both vCre and sCre are homologous proteins of Cre recombinase, but they show low homology with Cre and can specifically recognize vloxp and sloxp sites.


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