Inducible two-crossover dre recombinase system

Abstract

The application discloses a non-induction two-split Dre recombinase system, and belongs to the technical field of biology. The non-induction two-split Dre recombinase comprises the following forming steps: two-split design of Dre protein, screening of a two-split Dre protein combination with recombination enzyme activity without induction in 293T cells, and effective mediation of site-specific recombination of the two-split Dre protein in rox site stable 293T cells, mouse cell lines and E. coli. Two Dre peptide segments split between amino acids at Dre60 and 61 sites can form a recombination enzyme capable of mediating sequence recombination between rox sites with the assistance of an intein. Two Dre peptide segments split between amino acids at Dre191 and 192 sites can effectively complement each other without the assistance of any other fusion protein, mediate sequence recombination between rox sites, and cannot complement each other with split Cre peptide segments, so that when Dre and Cre are split at the same time, each can specifically mediate sequence recombination between the respective recognition sites.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a non-induced bisecting Dre recombinase system. Background Technology

[0002] Site-specific recombination systems are easy to operate and effective, and are widely used in synthetic biology, conditional knockout and knock-in, DNA inversion and translocation mutagenesis, and lineage tracing.

[0003] However, the application of site-specific recombinases is still subject to some technical limitations; for example, non-specific expression of the gene driving the recombinase leads to the same event occurring in other cell types; transient expression of the gene driving the recombinase in germ cells leads to gene knockout mice in conditional knockout mice, etc.

[0004] To better control the recombination of DNA in desired cells in both time and space, several strategies have been developed. For example, fusing Cre or other recombinases with mutated nuclear receptors or photoreactive proteins to construct inducible expression systems; combining different recombinases and their respective recognition sites in different ways; and splitting recombinases into two parts, each driven by a different promoter. However, these strategies still cannot meet all needs. For instance, when three or more proteins are required to specifically label a cell, these methods are insufficient. Therefore, more recombinase utilization strategies need to be developed. Summary of the Invention

[0005] 1. Technical problems to be solved

[0006] To address the problems existing in the prior art, the present invention aims to provide a non-induced split Dre recombinase, which enables two Dre peptides split between amino acids at Dre60 and 61 to form a recombinase that mediates sequence recombination between rox sites with the assistance of the intein peptide; and two Dre peptides split between amino acids at Dre191 and 192 can effectively complement each other without the assistance of any other fusion protein, mediating sequence recombination between rox sites. Furthermore, these two Dre peptides are not complementary to the split Cre peptides, ensuring that when Dre and Cre are split simultaneously, each specifically mediates sequence recombination between its respective recognition sites.

[0007] 2. Technical Solution

[0008] To solve the above problems, the present invention adopts the following technical solution.

[0009] The non-induced bisecting Dre recombinase comprises the following formation steps:

[0010] The bipartite design of S1 and Dre proteins;

[0011] Screening of bipartite Dre protein combinations with recombinase activity without induction in S2 and 293T cells;

[0012] S3, the bifid Dre protein can effectively mediate site-specific recombination in 293T cells that are stably transferred to the rox site;

[0013] S4, the bisecting Dre protein can effectively mediate site-specific recombination in mouse cell lines;

[0014] S5, the bifid Dre protein can effectively mediate site-specific recombination in prokaryotic cells E. coli;

[0015] S6. There is no complementary effect between the split Dre protein and the split Cre protein;

[0016] The two Dre peptides split between amino acids at positions 60 and 61 can form a recombinase that mediates sequence recombination between rox sites with the assistance of the intein peptide. The two Dre peptides split between amino acids 191 and 192 can effectively complement each other without the assistance of any other fusion protein, mediating sequence recombination between rox sites. Furthermore, these two Dre peptides are not complementary to the split Cre peptides, ensuring that when Dre and Cre are split simultaneously, each peptide specifically mediates sequence recombination between its respective recognition sites.

[0017] Furthermore, in S1, the dichotomy of Dre is selected at amino acids 60, 157, 191, 233, 245, and 282 of Dre. The dichotomy of Dre includes the N-terminal peptide segments: N60, N157, N191, N233, N245, and N282, and the C-terminal peptide segments: 61C, 158C, 192C, 234C, 246C, and 283C. The N-terminal peptide coding sequence of Dre has a translation stop codon added at the end, and the C-terminal peptide coding sequence of Dre has a translation start codon ATG added at the beginning.

[0018] Furthermore, the DNA coding sequences of each Dre peptide segment in S2 were ligated into the vector pcDNA3.1 / Myc-HisA. Each N-terminal peptide and C-terminal peptide was paired and combined with the pCAG-roxSTOProx-ZsGreen plasmid containing the same rox sequence, and then transformed into 293T cells. After 48 hours, the fluorescence intensity of each sample was detected. Compared with intact Dre, the recombinase activity of all the split Dre segments was reduced. Although the recombinase activity was lower than that of intact Dre, the N191 / 192C combination showed a certain recombinase activity. Cells expressing green fluorescence could be observed under a fluorescence microscope. However, when the N191 and 192C segments were combined with other peptide segments, they did not have recombinase activity, which also indicates that the split Dre peptide segments lose recombinase activity when they exist alone.

[0019] Furthermore, the bisection of the Dre protein in S3 requires Dre to cleave DNA in the cell nucleus, and the Dre cleavage simulation process includes:

[0020] a. A stable 293T cell line containing the rox site was constructed using lentivirus;

[0021] b. To improve detection sensitivity, two rox sequences with opposite orientations need to be added to both ends of the EGFP coding sequence in the cell. The orientation of the EGFP coding sequence is opposite to that of the promoter.

[0022] c. EGFP is expressed only after recombination occurs in the intermediate sequence at the rox site;

[0023] d. Add nuclear localization signal NLS to the N-terminus of the Dre N-terminal peptide and add nuclear localization signal NLS to the C-terminus of the C-terminal peptide;

[0024] e. To improve the activity of the peptide combination recombinase, the C-terminus of the DreN-terminal peptide is linked to the N-terminus of the DreN-terminal peptide, and the N-terminus of the C-terminal peptide is linked to the C-terminus of the DreN-terminal peptide.

[0025] By linking the coding sequence of the N-terminal peptide into a plasmid expressing the fluorescent protein mCherry, and the coding sequence of the C-terminal peptide into a plasmid expressing the fluorescent protein mTagBFP2, flow cytometry analysis was performed on cells exhibiting both red and blue coloration. The split N191 / 192C combination showed excellent protein complementarity without the need for any other accessory proteins, thus exercising recombinase activity. The presence or absence of the nuclear localization signal NLS at the C-terminus of 192C cells both indicated higher recombinase activity than the unsplit Dre. The intrin peptide Intein enabled the N60 / 61C combination, which lacked recombinase activity, to exhibit recombinase activity, and this activity was also stronger than that of the unsplit Dre.

[0026] Furthermore, the Dre protein in S4 is an effective peptide combination from S3. Both the N191 / 192C split combination and the N60 / 61C combination fused with the Intein peptide possess recombinase activity comparable to that of the unsplit Dre; the N191 / 192C split combination alone and the N60 / 61C combination fused with the Intein peptide can effectively mediate site-specific recombination in mouse cells.

[0027] Furthermore, in step S5, the LacZα coding sequence, which consists of two oppositely oriented rox sequences, is inserted into the prokaryotic plasmid pACYC177 at the PstⅠ site to construct the pACYC177-roxLacZα(rox) plasmid. In the pACYC177-roxLacZα(rox) plasmid, the inserted LacZα coding sequence is transcribed in the opposite direction to the promoter, and the inserted sequence disrupts the ampicillin resistance gene in the original plasmid. The bisected Dre peptide coding sequence is then ligated into pUC19, which can coexist with pACYC177 in E. coli. In the plasmid, to ensure the normal expression of both peptide segments, an SD sequence was added to the front of the coding sequence of each peptide segment. After co-transforming E. coli with the constructed plasmid containing the split Dre fragment and the pACYC177-roxLacZα(rox) plasmid, the β-galactosidase activity was detected. The N191 / 192C split combination showed recombinase activity comparable to that of the unsplit Dre, while the N60 / 61C combination did not have recombinase activity. These results indicate that the N191 / 192C split combination can still effectively mediate site-specific recombination in prokaryotic cells.

[0028] Furthermore, in step S6, the N191 / 192C bipartite combination is paired with the bipartite Cre protein, and their ability to cleave loxP and rox sequences is detected respectively. The bipartite combination of Cre protein N190 / 191C can mediate the recombination of loxP sites, and the bipartite combination of Dre protein N191 / 192C can mediate the recombination of rox sites. When the two are present in a crossover, neither loxP site recombination nor rox site recombination can be mediated.

[0029] Furthermore, the non-induced bisecting Dre recombinase system includes the non-induced bisecting Dre recombinase as described in claims 1-7.

[0030] 3. Beneficial effects

[0031] Compared with the prior art, the advantages of this invention are:

[0032] (1) In this scheme, the two Dre peptides split between the amino acids at Dre60 and 61 can form a recombinase that can mediate the recombination of sequences between rox sites with the help of the intein peptide; while the two Dre peptides split between the amino acids at Dre191 and 192 can effectively complement each other without the help of any other fusion protein, mediating the recombination of sequences between rox sites. Furthermore, these two Dre peptides and the split Cre peptides cannot complement each other, which can ensure that when Dre and Cre are split at the same time, each of them specifically mediates the recombination of sequences between their respective recognition sites. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the enzyme activity of the bipartite Dre in 293T cells according to the present invention (A: Dre bipartite scheme and peptide combination scheme; B: fluorescence value measurement results; C: cell fluorescence micrograph).

[0034] Figure 2 This is a schematic diagram of the enzyme activity of the bipartite Dre of the present invention in 293T cells stably transfected with the rox sequence (A: bipartite Dre reconstruction and combination scheme, nl represents N-terminal Intein, lc represents C-terminal Intein; B: proportion of cells expressing green fluorescent protein).

[0035] Figure 3 This is a schematic diagram of the enzyme activity of the bipartite Dre in mouse 4T1 cells (A: Bipartite Dre combination scheme, nl represents N-terminal Intein, lc represents C-terminal Intein; B: Proportion of cells expressing green fluorescent protein).

[0036] Figure 4 This is a schematic diagram of the enzyme activity of the bipartite Dre expression plasmid in E. coli according to the present invention (A: Construction scheme of the bipartite Dre expression plasmid; B: Detection results of β-galactosidase activity).

[0037] Figure 5 This is a schematic diagram showing that the bipartite Dr and bipartite Cre of the present invention cannot complement each other (A: combination scheme of bipartite Dr and bipartite Cre; B: proportion of cells expressing green fluorescence). Detailed Implementation

[0038] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0039] In the description of this invention, it should be noted that the terms "upper," "lower," "inner," "outer," "top / bottom," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0040] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installed," "equipped with," "sleeved / connected," "connected," etc., should be interpreted broadly. For example, "connection" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium; it can be a connection within two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0041] Example 1:

[0042] Please see Figure 1-5 The non-induced bisecting Dre recombinase comprises the following formation steps:

[0043] The bipartite design of S1 and Dre proteins;

[0044] Screening of bipartite Dre protein combinations with recombinase activity without induction in S2 and 293T cells;

[0045] S3, the bifid Dre protein can effectively mediate site-specific recombination in 293T cells that are stably transferred to the rox site;

[0046] S4, the bisecting Dre protein can effectively mediate site-specific recombination in mouse cell lines;

[0047] S5, the bifid Dre protein can effectively mediate site-specific recombination in prokaryotic cells E. coli;

[0048] S6. There is no complementary effect between the split Dre protein and the split Cre protein;

[0049] The two Dre peptides split between amino acids at positions 60 and 61 can form a recombinase that mediates sequence recombination between rox sites with the assistance of the intein peptide. The two Dre peptides split between amino acids 191 and 192 can effectively complement each other without the assistance of any other fusion protein, mediating sequence recombination between rox sites. Furthermore, these two Dre peptides are not complementary to the split Cre peptides, ensuring that when Dre and Cre are split simultaneously, each peptide specifically mediates sequence recombination between its respective recognition sites.

[0050] In S1, the dichotomy of Dre is chosen at amino acids 60, 157, 191, 233, 245, and 282. The dichotomy of Cre includes the N-terminal peptide of Dre: N60, N157, N191, N233, N245, and N282, and the C-terminal peptide of Dre: 61C, 158C, 192C, 234C, 246C, and 283C. The N-terminal peptide coding sequence of Dre has a translation stop codon added at the end, and the C-terminal peptide coding sequence of Dre has a translation start codon ATG added at the beginning.

[0051] The DNA coding sequences of each Dre peptide in S2 were ligated into the vector pcDNA3.1 / Myc-HisA. Each N-terminal peptide and each C-terminal peptide were paired and combined together with the pCAG-roxSTOProx-ZsGreen plasmid containing the same rox sequence, and then transformed into 293T cells. The fluorescence intensity of each sample was detected after 48 hours. Compared with intact Dre, the recombinase activity of all the split Dre peptides was reduced. Although the enzyme activity was lower than that of intact Dre, the N191 / 192C combination showed some recombinase activity. Cells expressing green fluorescence could be observed under a fluorescence microscope. However, when the N191 and 192C peptides were combined with other peptides, they did not have recombinase activity, which also indicates that the split Dre peptides lose recombinase activity when they exist alone.

[0052] The bifurcation of the Dre protein in S3 requires Dre to cut DNA in the cell nucleus. The Dre cutting process simulates the following:

[0053] a. A stable 293T cell line containing the rox site was constructed using lentivirus;

[0054] b. To improve detection sensitivity, two rox sequences with opposite orientations need to be added to both ends of the EGFP coding sequence in the cell. The orientation of the EGFP coding sequence is opposite to that of the promoter.

[0055] c. EGFP is expressed only after recombination occurs in the intermediate sequence at the rox site;

[0056] d. Add nuclear localization signal NLS to the N-terminus of the Dre N-terminal peptide and add nuclear localization signal NLS to the C-terminus of the C-terminal peptide;

[0057] e. To improve the activity of the peptide combination recombinase, the C-terminus of the DreN-terminal peptide is linked to the N-terminus of the DreN-terminal peptide, and the N-terminus of the C-terminal peptide is linked to the C-terminus of the DreN-terminal peptide.

[0058] By linking the coding sequence of the N-terminal peptide into a plasmid expressing the fluorescent protein mCherry, and the coding sequence of the C-terminal peptide into a plasmid expressing the fluorescent protein mTagBFP2, flow cytometry analysis showed that the split N191 / 192C combination exhibited excellent protein complementarity and exercised recombinase activity without the need for fusion of any other auxiliary proteins. The presence or absence of the nuclear localization signal NLS at the C-terminus of 192C resulted in higher recombinase activity than the unsplit Dre. The inteptide Intein enabled the N60 / 61C combination, which lacked recombinase activity, to exhibit recombinase activity, and this activity was also stronger than that of the unsplit Dre.

[0059] The Dre protein in S4 is an effective peptide combination from S3. Both the N191 / 192C split combination and the N60 / 61C combination fused with the Intein peptide possess recombinase activity comparable to the unsplit Dre; the N191 / 192C split combination alone and the N60 / 61C combination fused with the Intein peptide can effectively mediate site-specific recombination in mouse cells.

[0060] In S5, the LacZα coding sequence, wrapped by two oppositely oriented rox sequences, was inserted into the prokaryotic plasmid pACYC177 at the PstⅠ site to construct the pACYC177-roxLacZα(rox) plasmid. In the pACYC177-roxLacZα(rox) plasmid, the inserted LacZα coding sequence is opposite to the transcription direction of the promoter, and the inserted sequence disrupts the ampicillin resistance gene in the original plasmid. The bisected Dre peptide coding sequence was ligated into the pUC19 plasmid, which can coexist with pACYC177 in E. coli. To ensure proper expression of both peptide segments, an SD sequence was added to the front of the coding sequence of each peptide segment. After co-transforming E. coli with the constructed plasmid containing the split Dre fragment and the pACYC177-roxLacZα(rox) plasmid, β-galactosidase activity was detected. The N191 / 192C split combination exhibited recombinase activity comparable to the unsplit Dre fragment, while the N60 / 61C combination did not possess recombinase activity. These results indicate that the N191 / 192C split combination can still effectively mediate site-specific recombination in prokaryotic cells.

[0061] In S6, the N191 / 192C bipartite combination was paired with the bipartite Cre protein to test their ability to cleave loxP and rox sequences, respectively. The Cre protein bipartite combination N190 / 191C can mediate loxP site recombination, and the Dre protein bipartite combination N191 / 192C can mediate rox site recombination. When the two are present in a crossover, neither loxP site recombination nor rox site recombination can be mediated.

[0062] The non-inducible di-Dre recombinase system includes the non-inducible di-Dre recombinase as described in claims 1-7.

[0063] The above description is merely a preferred embodiment of the present invention; however, the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and its improved concept, should be covered within the scope of protection of the present invention.

Claims

1. A non-inducible bisecting Dre recombinase, characterized in that: It consists of an N-terminal peptide and a C-terminal peptide of the Dre protein; the Dre protein is split between amino acids 191 and 192, the N-terminal peptide is the segment of amino acids 1-191 of the Dre protein, and the C-terminal peptide is 192C starting from amino acid 192; the N-terminal peptide and the C-terminal peptide spontaneously complement each other without the need for any fusion protein, restoring recombinase activity, specifically recognizing and mediating sequence recombination between rox sites, the N-terminal peptide is connected to a nuclear localization signal (NLS), and the C-terminal peptide is connected to a nuclear localization signal (NLS).

2. The non-induced bisecting Dre recombinase according to claim 1, characterized in that: The N-terminal and C-terminal peptides effectively mediate site-specific recombination in mammalian cells; the mammalian cells are stable 293T cells containing rox sites or mouse cell lines.

3. The non-induced bisecting Dre recombinase according to claim 1, characterized in that: The N-terminal and C-terminal peptides effectively mediate site-specific recombination in E. coli prokaryotic cells.

4. The non-induced bisecting Dre recombinase according to claim 1, characterized in that: The bisecting Dre recombinase and the bisecting Cre recombinase do not have a cross-complementary effect; when they coexist, they specifically mediate sequence recombination between their corresponding recognition sites.

5. A non-induced bisecting Dre recombinase system, characterized in that: Including the non-induced bisecting Dre recombinase as described in any one of claims 1-4.

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