CN111705079B

CN111705079B - Gene editing method based on Vcre-Vloxp recombinase system and application thereof

Info

Publication number: CN111705079B
Application number: CN202010840799.0A
Authority: CN
Inventors: 赵金龙; 朱石磊; 李雯婧; 林梓凡
Original assignee: Gempharmatech Co ltd
Current assignee: Shanghai Yaokang Biotechnology Co.,Ltd.
Priority date: 2020-08-20
Filing date: 2020-08-20
Publication date: 2020-11-20
Anticipated expiration: 2040-08-20
Also published as: CN111705079A

Abstract

The invention provides a gene editing method based on a Vcre-Vloxp recombinase system and application thereof, wherein the method comprises the following steps: (1) ligating a Vloxp sequence to the 5 'end or 3' end of the double-stranded DNA, or inserting said Vloxp sequence between functional regions of the double-stranded DNA; (2) and then mixing the double-stranded DNA, the Vcre protein and the reaction solution for gene editing, and transferring the mixture into a target cell to finish the gene editing. When the phenomenon of repeated connection of double-stranded DNA occurs in the integration process, the double-stranded DNA is sheared under the action of Vcre protein, only one double-stranded DNA is finally left, and no matter how many double-stranded DNAs are connected in series, the Vcre protein can cut all the repeated fragments from the first Vloxp sequence to the last Vloxp sequence, so that the method can obviously reduce the phenomenon of repeated connection of recombinant DNA fragments in the integration process.

Description

Gene editing method based on Vcre-Vloxp recombinase system and application thereof

Technical Field

The invention relates to the field of genetic engineering, and relates to a gene editing method based on a Vcre-Vloxp recombinase system and application thereof.

Background

The Cre-Loxp recombinase system mainly consists of site-specific Cre recombinase and LoxP sequences, and is firstly discovered from P1 phage by Steinberg and the like. Wherein, the Cre recombinase is a monomeric protein which is coded by P1 bacteriophage Cre gene and has 343 amino acids, and belongs to lambda Int enzyme supergene family. It not only has catalytic activity, but also can recognize specific DNA sequence, i.e. LoxP (focus of X (cross) -ovorinP 1) site, similar to restriction enzyme. LoxP consists of two 13 bp inverted repeat sequences and a middle 8 bp spacer sequence, the 8 bp spacer sequence also determines the orientation of the LoxP, a gene sequence between LoxP sites is deleted or recombined by Cre recombinase, and the 13 bp inverted repeat sequence is a binding domain of Cre enzyme.

The recombination enzyme systems of Vcre-VloxP and SCre-SloxP are two systems developed on the basis of a Cre-Loxp recombination enzyme system, but the similarity of Vcre and SCre and Cre enzyme is very low, and the homology of amino acid sequences of the Vcre-VloxP and SCre-SloxP recombination enzyme systems is 29% and 31% respectively. Thus, although their recognition sites are similar to the structure of 13-8-13 of Loxp, they differ from Loxp; further studies showed no cross-reaction between Vcre-VloxP, SCRe-SloxP and Cre-Loxp systems (see Minorikawa S, Nakayama M. recombined cassette exchange (RMCE) and BAC engineering via Vcre/VloxP and SCRe/SloxP systems Biotechnology. 2011;50(4):235-246. doi: 10.2144/000113649.).

Technologies such as ZFN, TALEN, CRISPR-Cas9 and the like realize gene editing by causing double-stranded DNA break of an editing site, and have been widely applied to precise gene editing of various model organisms including mice and human beings. And with the intensive research and interpretation of two repair modes, Non-Homologous end joining (NHEJ) and Homologous Recombination (HR), the DNA repair mechanism is gradually applied to the gene editing technology for directionally transforming genes. Researchers use nuclease technology to purposefully break the double strand of DNA, and use this mechanism to regulate the expression of a target gene or introduce a selectable marker.

The ZFN, TALEN and CRISPR-Cas9 proteins cut DNA Double strands to form DNA Double Strand Breaks (DSBs), a NHEJ repair mechanism is utilized, random deletion or base insertion is carried out to cause frameshift mutation, and the target gene is knocked out; introducing a section of exogenous gene while shearing, and accessing the exogenous gene into DSBs sites by NHEJ to realize gene insertion; or the two ends are respectively provided with a shearing site, the middle segment is free, and the two ends are connected through an NHEJ mechanism to form large-segment gene knockout. Meanwhile, a foreign gene and a homologous sequence are added during shearing, and the gene can be accurately knocked in and replaced at a fixed point under the action of an HR mechanism. The method is widely used in the biomedical field at present, so that the editing is more efficient and the sequence is more specific.

Nevertheless, this technique still has drawbacks. Researches show that in the process of establishing six knockout mouse models under different conditions, the donor DNA template has multiple unnecessary head-tail tandem phenomena. In most cases, these multiple integration events are not recognized by conventional PCR analysis, which seriously affects the accuracy of the technique (see Skryabin BV, et al. Pervasive head-to-tail insertions of DNA templates determined CRISPR-Cas9-mediated genome editing events Sci adv. 2020;6(7): eaax 2941.).

In the gene targeting process, the recombinant DNA fragments have a head-to-tail tandem phenomenon, and the tandem connection causes the failure of gene targeting. In the field, Southern, qPCR, PCR and other methods are commonly used to detect whether there is a tandem, but no effective method for reducing the tandem has been found.

Therefore, how to reduce the tandem repeat phenomenon of the recombinant DNA fragment by using the conditional knockout element has important significance on the development and perfection of gene editing technology.

Disclosure of Invention

In view of the problems in the prior art, the invention provides a gene editing method based on a Vcre-Vloxp recombinase system and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a gene editing method based on a Vcre-Vloxp recombinase system, which specifically comprises the following steps:

(1) ligating a Vloxp sequence to the 5 'end or 3' end of the double-stranded DNA, or inserting said Vloxp sequence between functional regions of the double-stranded DNA;

the Vloxp sequence is a nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO. 2;

(2) mixing the double-stranded DNA, the Vcre protein and the reaction solution for gene editing, and transferring the mixture into a target cell to finish gene editing and removal of tandem fragments;

the Vcre protein has an amino acid sequence shown as SEQ ID NO. 3;

the double-stranded DNA also comprises any one or the combination of at least two of a Loxp sequence, a Loxp mutant sequence, a Frt sequence or a Rox sequence;

in the invention, in order to reduce the phenomenon of multi-copy and tandem duplication when the exogenous gene is integrated into a chromosome and improve the controllability of the integration, a conditional gene knockout (CKO) element Vloxp is inserted into the exogenous gene, namely a double-stranded DNA fragment. When the target gene is repeatedly connected, the target gene is sheared under the action of Vcre protein, and only one double-stranded DNA is finally left. And regardless of how many double-stranded DNAs are concatenated, Vcre protein can excise all the repetitive fragments from the first Vloxp sequence to the last Vloxp sequence, leaving only one copy of double-stranded DNA.

Meanwhile, the double-stranded DNA comprises any one or at least two of a Loxp sequence, a Frt sequence or a Rox sequence, namely, for a cell which needs to be conditionally expressed or knocked out by using a recombinase system such as Cre-Loxp, Flp-Frt and Dre-Rox, the invention can simultaneously insert a pair of Loxp sequence, Frt sequence or Rox sequence into the double-stranded DNA. The selected Vcre-Vloxp system does not influence the normal gene editing step, and does not generate cross influence on the functions of other recombinase systems.

In addition, the difference between the invention and the common CKO is that: in order to realize gene knockout, CKO needs to insert 2 Loxp sequences into one fragment, or insert 1 Loxp sequence into two different fragments; in the invention, only 1 Vloxp sequence is inserted into one fragment for the purpose of reducing the tandem repeat phenomenon. Meanwhile, the DNA segment with Vloxp is introduced into cells together with Vcre, while CKO and the like realize genome recombination of Loxp firstly and then introduce Cre protein to realize gene editing.

Meanwhile, in the present invention, the insertion position of the Vloxp sequence is limited, and it is preferable that the insertion position does not affect the expression and translation, such as the two ends of the fragment or between the functional regions. Here, it should be noted that the functional regions may be, for example: promoter, cDNA or PloyA gene elements. Among them, the placement in the protein coding sequence is a second choice, and if necessary, two bases need to be added on the Vloxp sequence so as not to affect the gene transcription and translation.

In the present invention, SEQ ID NO.1 is as follows:

TCAATTTCTGAGAACTGTCATTCTCGGAAATTGA；

SEQ ID NO.2 is shown below:

TCAATTTCTGAGAACTGTCATTCTCGGAAATTGATG。

SEQ ID NO.3 is shown below:

MPKKKRKVIENQLSLLGDFSGVRPDDVKTAIQAAQKKGINVAENEQFKAAFEHLLNEFKKREERYSPNTLRRLESAWTCFVDWCLANHRHSLPATPDTVEAFFIERAEELHRNTLSVYRWAISRVHRVAGCPDPCLDIYVEDRLKAIARKKVREGEAVKQASPFNEQHLLKLTSLWYRSDKLLLRRNLALLAVAYESMLRASELANIRVSDMELAGDGTAILTIPITKTNHSGEPDTCILSQDVVSLLMDYTEAGKLDMSSDGFLFVGVSKHNTCIKPKKDKQTGEVLHKPITTKTVEGVFYSAWETLDLGRQGVKPFTAHSARVGAAQDLLKKGYNTLQIQQSGRWSSGAMVARYGRAILARDGAMAHSRVKTRSAPMQWGKDEKD。

preferably, the nucleotide sequence for encoding the Vcre protein is shown as SEQ ID NO. 4.

SEQ ID NO.4 is shown below:

ATGCCCAAGAAAAAGCGGAAAGTGATCGAGAACCAGCTGAGCCTGCTGGGCGACTTTTCTGGCGTGCGGCCCGACGATGTGAAAACCGCCATTCAGGCCGCCCAGAAAAAGGGCATCAACGTGGCCGAGAACGAGCAGTTCAAGGCCGCCTTCGAGCATCTGCTGAACGAGTTCAAGAAGCGGGAAGAGAGATACAGCCCCAACACCCTGCGGCGGCTGGAAAGCGCCTGGACCTGCTTCGTGGATTGGTGCCTGGCCAACCACAGACACAGCCTGCCTGCCACCCCCGATACCGTGGAAGCCTTCTTCATCGAGCGGGCCGAGGAACTGCACCGGAACACCCTGAGCGTGTACAGATGGGCCATCAGCCGGGTGCACAGAGTGGCCGGATGCCCTGATCCCTGCCTGGACATCTACGTGGAAGATCGGCTGAAGGCCATTGCCCGGAAGAAAGTGCGGGAAGGCGAGGCCGTGAAGCAGGCCAGCCCTTTCAACGAGCAGCATCTGCTGAAGCTGACCAGCCTGTGGTACAGAAGCGACAAGCTGCTGCTGCGGCGGAACCTGGCTCTGCTGGCTGTGGCCTACGAGAGCATGCTGAGAGCCAGCGAGCTGGCCAACATCCGGGTGTCCGATATGGAACTGGCCGGCGACGGAACCGCCATCCTGACCATCCCTATCACCAAGACCAACCACTCCGGCGAGCCCGATACCTGCATCCTGTCCCAGGATGTGGTGTCCCTGCTGATGGACTACACCGAGGCCGGCAAGCTGGATATGAGCAGCGACGGCTTCCTGTTCGTGGGCGTGTCCAAGCACAACACCTGTATCAAGCCCAAGAAGGACAAGCAGACCGGCGAGGTGCTGCACAAGCCCATCACCACCAAGACAGTGGAAGGCGTGTTCTACAGCGCCTGGGAGACACTGGACCTGGGCAGACAGGGCGTGAAGCCTTTCACAGCCCACAGCGCCAGAGTGGGAGCCGCTCAGGACCTGCTGAAGAAGGGCTACAATACCCTGCAGATCCAGCAGTCCGGCCGGTGGTCTAGCGGAGCCATGGTGGCCAGATACGGCAGAGCCATCCTGGCTAGGGATGGCGCTATGGCCCACAGCAGAGTGAAAACCAGATCCGCCCCCATGCAGTGGGGCAAGGACGAGAAGGACTGA。

preferably, the Vcre protein is synthesized by Escherichia coli engineering bacteria expressing the Vcre protein.

Preferably, the Vloxp sequence is ligated to the 3' end of the double stranded DNA.

As a preferred embodiment of the present invention, the working concentration of the double-stranded DNA in the step (2) is 2 to 10 ng/. mu.L, and may be, for example, 2 ng/. mu.L, 3 ng/. mu.L, 4 ng/. mu.L, 5 ng/. mu.L, 6 ng/. mu.L, 7 ng/. mu.L, 8 ng/. mu.L, 9 ng/. mu.L, or 10 ng/. mu.L.

Preferably, the working concentration of the Vcre protein in the step (2) is 80-120 ng/μ L, such as 80 ng/μ L, 85 ng/μ L, 90 ng/μ L, 95 ng/μ L, 100 ng/μ L, 105 ng/μ L, 110 ng/μ L, 115 ng/μ L or 120 ng/μ L.

In the invention, the reaction solution for gene editing can be the reaction solution required by any one of gene editing methods such as CRISPR-Cas9 gene editing, TALEN gene editing, ZFN gene editing, ES homologous recombination or TG transgenosis and the like.

Preferably, the method for transferring the mixed reaction solution into the target cell in step (2) comprises a prokaryotic microinjection method. Preferably, the cell of interest is a mammalian cell.

As a preferred embodiment of the present invention, the gene editing method comprises the steps of:

(2) mixing the double-stranded DNA, the Vcre protein and the reaction solution for gene editing, and injecting the mixed reaction solution into a target cell by using a prokaryotic microinjection method to complete gene editing and removal of the tandem fragments;

the working concentration of the Vcre protein is 80-120 ng/mu L, and the working concentration of the double-stranded DNA is 2-10 ng/mu L;

the Vcre protein has an amino acid sequence shown as SEQ ID NO. 3;

the double-stranded DNA further comprises any one or a combination of at least two of a Loxp sequence, a Loxp mutant sequence, a Frt sequence and a Rox sequence.

In a second aspect, the use of a method of gene editing as described in the first aspect for gene knock-out, gene mutation or site-directed integration of a gene.

The recitation of numerical ranges herein includes not only the above-recited values, but also any values between any of the above-recited numerical ranges not recited, and for brevity and clarity, is not intended to be exhaustive of the specific values encompassed within the range.

Compared with the prior art, the invention has at least the following beneficial effects:

in the invention, a Vloxp sequence is inserted between the 5 'end, the 3' end or the functional region of the double-stranded DNA, and then mixed with a reaction system for editing Vcre protein and genes under the condition of not influencing the normal function of the double-stranded DNA; compared with other conditional gene knockout systems, the Vcre-Vloxp system has the best efficiency of reducing the tandem connection;

meanwhile, the Cre protein and the Vloxp sequence as well as the Vcre protein and the Loxp sequence have no cross reaction, so that the method is an ideal redundant copy deletion original. By applying the Vloxp-Vcre system, the invention can effectively reduce the tandem rate, reduce the gene tandem rate from 47.6% to 5.9%, and improve the success rate of targeting.

Drawings

FIG. 1 is a schematic diagram of the structure of the double-stranded DNA fragment constructed in example 1.

Detailed Description

The technical solutions of the present invention are further described in the following embodiments with reference to the drawings, but the following examples are only simple examples of the present invention and do not represent or limit the scope of the present invention, which is defined by the claims.

In the following examples, female (C57 BL/6JGpt, 4 weeks old), male (C57 BL/6JGpt, >8 weeks old) mice: all produced by Jiangsu Jiejiaokang Biotech limited;

in the following examples, reagents used included: hyaluronidase working solution, DPBS working solution, injection buffer solution, PMSG/HCG working solution, M16, M2, 75% alcohol and mineral oil;

in the following examples, the apparatus used included: inverted microscope, micro-operation system, micro sample loader, needle drawing instrument, needle forging instrument, and CO₂Incubator, refrigerator, stereomicroscope;

in the following examples, consumables were used which included: GD-1 glass needle, microsyringe, 3.5 cm dish, fixed tube, fallopian tube, 1mL syringe, surgical instrument, etc.; are all commercially available from conventional sources.

In the following examples, the remaining steps can be performed by means of techniques commonly used in the art, such as digestion, purification, etc., except for specific descriptions.

Example 1

This example provides a gene editing method based on the Vcre-Vloxp recombinase system.

(1) Construction of double stranded DNA (dsDNA):

the structure of dsDNA constructed in this example is schematically shown in FIG. 1.

The dsDNA is shown in SEQ ID NO.5, wherein CMV Promoter is adopted as Promoter (Promoter) and BGHpA Terminator is adopted as Terminator (Terminator). Therefore, dsDNA can be marked as CMV-Loxp-5 × stop-Loxp-EGFP-Vloxp-BGHpA.

Constructing the designed sequence on a plasmid, and performing enzyme digestion and purification to obtain dsDNA;

after completion of the digestion, the dsDNA was recovered by purification and diluted to 5 ng/. mu.L with TE buffer.

SEQ ID NO.5 is shown below:

CGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGATAACTTCGTATAGCATACATTATACGAAGTTATCCTCAGCACCATGGCTAGCGGCAGCCTCGGAGTTTGAATAGATAGAATAAAATATCTTTATTTTCATTCCATCTGTGTGTTGGTTTTTTGTGTGAGATCTACGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGGCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGGTCTCCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCCCTTCCCTGTCCTTCTGCCTCAGCTAATTGAGTAGGGGGGAGGCTAACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGCAATAAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTCATAAACGCGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCATTGGGGCCAATACGCCCGCGTTTCTTCCTTTTCCCCACCCCACCCCCCAAGTTCGGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAGGCCGTGGAATTCGTAAATGAATTTTCTGTATGAGGTCGCGATGAATAAATGAAAGCTTGCAGATCTGCGACTCTAGAGGATCTGCGACTCTAGAGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTGCGACTCTAGAGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTGCGACTCTAGAGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCCCCATCAAGCTGATCCGGAACCCTTAATATAACTTCGTATAGCATACATTATACGAAGTTATAGACCCAAGCTTGGTACCGAGCTCGGATCCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAATCAATTTCTGAGAACTGTCATTCTCGGAAATTGAGATATCCATCACACTGGCGGCCGCTCGAGCATGCATCTAGAGGGCCCTATTCTATAGTGTCACCTAAATGCTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGA。

(2) microinjection of mouse fertilized egg

Injecting dsDNA, Vcre protein and a reaction solution for gene editing into fertilized eggs of a mouse by a conventional injection method, wherein the number of the injected fertilized eggs is 400;

in the embodiment, the used reaction solution for gene editing contains a CRISPR-Cas9 reaction system, wherein the working concentration of sgRNA is 5 ng/μ L, and the working concentration of Cas9 mRNA is 10 ng/μ L;

the concentration of the double-stranded DNA mixed with the CRISPR-Cas9 reaction system is 2 ng/mu L; the concentration of the mixed Vcre protein and CRISPR-Cas9 reaction system is 80 ng/mu L;

(3) transplantation, breeding and identification

Transplanting the fertilized ovum into oviduct of surrogate mouse, and after about 20 days, F0 mouse is born; after one week of birth, the tail is cut, the rat tail genome is extracted, PCR and sequencing identification are carried out, the tandem rate is counted,

finally, the number of injected fertilized eggs is 400, the number of young animals is 56, the number of positive PCR detection is 17, only 1 mouse is connected in series, and the connection rate is 5.9%.

Comparative example 1

The difference from example 1 is that in this comparative example, Vcre protein was not added, the remaining steps are in accordance with example 1,

finally, the number of injected fertilized eggs is 400, the number of piglets is 64, the number of positive PCR detection is 21, 10 mice are connected in series, and the series rate is 47.6%.

In conclusion, the VloxP is added into the dsDNA, and the Vcre protein is added into the injection system, so that the series connection proportion can be greatly reduced, and the series connection rate can be reduced to 5.9% from 47.6%; meanwhile, for cells which are subjected to conditional expression or knockout by using or need to be subjected to conditional expression by using a recombinase system such as Cre-Loxp, Flp-Frt and Dre-Rox, the gene editing method based on the Vcre-Vloxp recombinase system provided by the invention does not influence the normal gene editing step.

The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.

SEQUENCE LISTING

<110> Jiangsu Jiejiaokang Biotech limited

<120> gene editing method based on Vcre-Vloxp recombinase system and application thereof

<130> 20200807

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 34

<212> DNA

<213> Artificial Synthesis

<400> 1

tcaatttctg agaactgtca ttctcggaaa ttga 34

<210> 2

<211> 36

<212> DNA

<213> Artificial Synthesis

<400> 2

tcaatttctg agaactgtca ttctcggaaa ttgatg 36

<210> 3

<211> 387

<212> PRT

<213> Artificial Synthesis

<400> 3

Met Pro Lys Lys Lys Arg Lys Val Ile Glu Asn Gln Leu Ser Leu Leu

1 5 10 15

Gly Asp Phe Ser Gly Val Arg Pro Asp Asp Val Lys Thr Ala Ile Gln

20 25 30

Ala Ala Gln Lys Lys Gly Ile Asn Val Ala Glu Asn Glu Gln Phe Lys

35 40 45

Ala Ala Phe Glu His Leu Leu Asn Glu Phe Lys Lys Arg Glu Glu Arg

50 55 60

Tyr Ser Pro Asn Thr Leu Arg Arg Leu Glu Ser Ala Trp Thr Cys Phe

65 70 75 80

Val Asp Trp Cys Leu Ala Asn His Arg His Ser Leu Pro Ala Thr Pro

85 90 95

Asp Thr Val Glu Ala Phe Phe Ile Glu Arg Ala Glu Glu Leu His Arg

100 105 110

Asn Thr Leu Ser Val Tyr Arg Trp Ala Ile Ser Arg Val His Arg Val

115 120 125

Ala Gly Cys Pro Asp Pro Cys Leu Asp Ile Tyr Val Glu Asp Arg Leu

130 135 140

Lys Ala Ile Ala Arg Lys Lys Val Arg Glu Gly Glu Ala Val Lys Gln

145 150 155 160

Ala Ser Pro Phe Asn Glu Gln His Leu Leu Lys Leu Thr Ser Leu Trp

165 170 175

Tyr Arg Ser Asp Lys Leu Leu Leu Arg Arg Asn Leu Ala Leu Leu Ala

180 185 190

Val Ala Tyr Glu Ser Met Leu Arg Ala Ser Glu Leu Ala Asn Ile Arg

195 200 205

Val Ser Asp Met Glu Leu Ala Gly Asp Gly Thr Ala Ile Leu Thr Ile

210 215 220

Pro Ile Thr Lys Thr Asn His Ser Gly Glu Pro Asp Thr Cys Ile Leu

225 230 235 240

Ser Gln Asp Val Val Ser Leu Leu Met Asp Tyr Thr Glu Ala Gly Lys

245 250 255

Leu Asp Met Ser Ser Asp Gly Phe Leu Phe Val Gly Val Ser Lys His

260 265 270

Asn Thr Cys Ile Lys Pro Lys Lys Asp Lys Gln Thr Gly Glu Val Leu

275 280 285

His Lys Pro Ile Thr Thr Lys Thr Val Glu Gly Val Phe Tyr Ser Ala

290 295 300

Trp Glu Thr Leu Asp Leu Gly Arg Gln Gly Val Lys Pro Phe Thr Ala

305 310 315 320

His Ser Ala Arg Val Gly Ala Ala Gln Asp Leu Leu Lys Lys Gly Tyr

325 330 335

Asn Thr Leu Gln Ile Gln Gln Ser Gly Arg Trp Ser Ser Gly Ala Met

340 345 350

Val Ala Arg Tyr Gly Arg Ala Ile Leu Ala Arg Asp Gly Ala Met Ala

355 360 365

His Ser Arg Val Lys Thr Arg Ser Ala Pro Met Gln Trp Gly Lys Asp

370 375 380

Glu Lys Asp

385

<210> 4

<211> 1164

<212> DNA

<213> Artificial Synthesis

<400> 4

atgcccaaga aaaagcggaa agtgatcgag aaccagctga gcctgctggg cgacttttct 60

ggcgtgcggc ccgacgatgt gaaaaccgcc attcaggccg cccagaaaaa gggcatcaac 120

gtggccgaga acgagcagtt caaggccgcc ttcgagcatc tgctgaacga gttcaagaag 180

cgggaagaga gatacagccc caacaccctg cggcggctgg aaagcgcctg gacctgcttc 240

gtggattggt gcctggccaa ccacagacac agcctgcctg ccacccccga taccgtggaa 300

gccttcttca tcgagcgggc cgaggaactg caccggaaca ccctgagcgt gtacagatgg 360

gccatcagcc gggtgcacag agtggccgga tgccctgatc cctgcctgga catctacgtg 420

gaagatcggc tgaaggccat tgcccggaag aaagtgcggg aaggcgaggc cgtgaagcag 480

gccagccctt tcaacgagca gcatctgctg aagctgacca gcctgtggta cagaagcgac 540

aagctgctgc tgcggcggaa cctggctctg ctggctgtgg cctacgagag catgctgaga 600

gccagcgagc tggccaacat ccgggtgtcc gatatggaac tggccggcga cggaaccgcc 660

atcctgacca tccctatcac caagaccaac cactccggcg agcccgatac ctgcatcctg 720

tcccaggatg tggtgtccct gctgatggac tacaccgagg ccggcaagct ggatatgagc 780

agcgacggct tcctgttcgt gggcgtgtcc aagcacaaca cctgtatcaa gcccaagaag 840

gacaagcaga ccggcgaggt gctgcacaag cccatcacca ccaagacagt ggaaggcgtg 900

ttctacagcg cctgggagac actggacctg ggcagacagg gcgtgaagcc tttcacagcc 960

cacagcgcca gagtgggagc cgctcaggac ctgctgaaga agggctacaa taccctgcag 1020

atccagcagt ccggccggtg gtctagcgga gccatggtgg ccagatacgg cagagccatc 1080

ctggctaggg atggcgctat ggcccacagc agagtgaaaa ccagatccgc ccccatgcag 1140

tggggcaagg acgagaagga ctga 1164

<210> 5

<211> 3559

<212> DNA

<213> Artificial Synthesis

<400> 5

cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc 60

aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt 120

aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta 180

tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg actatttacg 240

gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga 300

cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt 360

tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg 420

gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc 480

cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg 540

taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat 600

aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatac 660

gactcactat agggataact tcgtatagca tacattatac gaagttatcc tcagcaccat 720

ggctagcggc agcctcggag tttgaataga tagaataaaa tatctttatt ttcattccat 780

ctgtgtgttg gttttttgtg tgagatctac gggtggcatc cctgtgaccc ctccccagtg 840

cctctcctgg ccctggaagt tgccactcca gtgcccacca gccttgtcct aataaaatta 900

agttgcatca ttttgtctga ctaggtgtcc ttctataata ttatggggtg gaggggggtg 960

gtatggagca aggggcaagt tgggaagaca acctgtaggg cctgcggggt ctattgggaa 1020

ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa 1080

gcgattctcc tgcctcaggc tcccgagttg ttgggattcc aggcatgcat gaccaggctc 1140

agctaatttt tgtttttttg gtagagacgg ggtttcacca tattggccag gctggtctcc 1200

aactcctaat ctcaggtgat ctacccacct tggcctccca aattgctggg attacaggcg 1260

tgaaccactg ctcccttccc tgtccttctg cctcagctaa ttgagtaggg gggaggctaa 1320

ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac 1380

agaataaaac gcacgggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc 1440

tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc 1500

ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg 1560

gcggcaggcc gtggaattcg taaatgaatt ttctgtatga ggtcgcgatg aataaatgaa 1620

agcttgcaga tctgcgactc tagaggatct gcgactctag aggatcataa tcagccatac 1680

cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa 1740

acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa 1800

ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg 1860

tggtttgtcc aaactcatca atgtatctta tcatgtctgg atctgcgact ctagaggatc 1920

ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc 1980

cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct 2040

tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca 2100

ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatctgc 2160

gactctagag gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa 2220

acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact 2280

tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata 2340

aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc 2400

atgtctggat ccccatcaag ctgatccgga acccttaata taacttcgta tagcatacat 2460

tatacgaagt tatagaccca agcttggtac cgagctcgga tccatggtga gcaagggcga 2520

ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca 2580

caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa 2640

gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac 2700

ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa 2760

gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa 2820

ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct 2880

gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta 2940

caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt 3000

caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa 3060

cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc 3120

cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac 3180

cgccgccggg atcactctcg gcatggacga gctgtacaag taatcaattt ctgagaactg 3240

tcattctcgg aaattgagat atccatcaca ctggcggccg ctcgagcatg catctagagg 3300

gccctattct atagtgtcac ctaaatgcta gagctcgctg atcagcctcg actgtgcctt 3360

ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg 3420

ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt 3480

gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca 3540

atagcaggca tgctgggga 3559

Claims

1. A gene editing method based on a Vcre-Vloxp recombinase system is characterized by comprising the following steps:

the Vcre protein has an amino acid sequence shown as SEQ ID NO. 3;

2. The gene editing method of claim 1, wherein the nucleotide sequence encoding the Vcre protein is shown in SEQ ID No. 4.

3. The gene editing method of claim 2, wherein the Vcre protein is synthesized from an engineered escherichia coli strain expressing Vcre protein.

4. The method for gene editing according to claim 1, wherein the Vloxp sequence in step (1) is ligated to the 3' end of the double-stranded DNA.

5. The gene editing method of claim 1, wherein the working concentration of Vcre protein in step (2) is 80-120 ng/μ L.

6. The method of gene editing according to claim 1, wherein the working concentration of the double-stranded DNA in step (2) is 2 to 10 ng/. mu.L.

7. The method for gene editing according to claim 1, wherein the method for transferring the mixed reaction solution into the target cell in step (2) comprises a prokaryotic microinjection method.

8. The method for gene editing according to claim 7, wherein the target cell in step (2) is a mammalian cell.

9. The gene editing method according to claim 1, comprising the steps of:

(2) mixing the double-stranded DNA, the Vcre protein and the gene editing system, and injecting the mixed reaction solution into a target cell by using a prokaryotic microinjection method to complete gene editing and removal of the tandem segment;

the Vcre protein has an amino acid sequence shown as SEQ ID NO. 3;

10. Use of a method of gene editing according to any one of claims 1 to 9 for gene knockout, gene mutation or gene site-directed integration.