阅读说明：本技术 一种真核生物CRISPR-Cas9双gRNA载体及构建方法 (Eukaryotic organism CRISPR-Cas9 double gRNA vector and construction method thereof ) 是由 马三垣 常珈菘 夏庆友 于 2020-05-07 设计创作，主要内容包括：本发明涉及一种真核生物CRISPR-Cas9双gRNA载体及构建方法,以piggyBac转座子系统为递送系统,以酶切连接的方法构建双gRNA载体,其特征在于,借助piggyBac转座子系统超大的承载能力和广泛的物种有效性,Cas9表达框和双gRNA表达框都在同一个载体上,能够高效简便的在众多真核生物中实现双gRNA同时发挥作用,实现两个基因同时敲除或者功能基因片段(编码基因、非编码RNA、基因组功能元件等)删除等。(The invention relates to a eukaryotic organism CRISPR-Cas9 double gRNA vector and a construction method thereof, wherein a piggyBac transposon system is used as a delivery system, and a double gRNA vector is constructed by an enzyme digestion connection method.)

1. A construction method of a double gRNA vector of eukaryotic organism CRISPR-Cas9 is characterized by comprising the following specific steps:

(1) constructing a piggyBac transposon system mediated eukaryote CRISPR-Cas9 double gRNA framework vector, namely pB-CRISPR, wherein the nucleotide sequence of the vector is shown as SEQ ID NO. 1; the gene element delivery system of the vector is a piggyBac transposon system, and the gene knockout system is a CRISPR/Cas9 system;

(2) constructing a template vector for providing sgRNA scaffold and U6 promoters, namely T-DGP-7, wherein the nucleotide sequence of the template vector is shown as SEQ ID NO. 2;

(3) designing a targeting site, constructing a primer pair of a double gRNA vector, and then performing PCR amplification by using the primer pair with the T-DGP-7 obtained in the step (2) as a template to obtain an amplification product named as PCR-DGP 7-XY;

(4) digesting the pB-CRISPR obtained in the step (1) by using an endonuclease AarI as a framework, digesting the PCR-DGP7-XY obtained in the step (3) by using BbsI as a fragment, and connecting the framework and the fragment to form a double gRNA vector which is named as pB-Dul-CRISPR-XY;

(5) and (3) mixing the pB-Dul-CRISPR-XY obtained in the step (4) with a piggyBactransposon expression vector A3-helper with the nucleotide sequence shown as SEQ ID NO.3 for transfection of eukaryotic cells, and screening to obtain the vector.

2. The construction method according to claim 1, wherein the specific method of step (1) is as follows:

(1-1) synthesizing a vector PUC57-IE2-Zeocin-Ser1PA containing a Zeocin resistance gene expression cassette, wherein the nucleotide sequence of the vector is shown as SEQ ID NO. 6;

(1-2) connecting a Zeocin resistance gene expression frame IE2-Zeocin-Ser1PA on a vector PUC57-IE2-Zeocin-Ser1PA to a piggyBac transposon basic vector piggyBacModify with a nucleotide sequence shown as SEQ ID No.7 to construct an intermediate vector pB-Modified { IE2-Zeocin-Ser1PA }, wherein the nucleotide sequence is shown as SEQ ID No. 8;

(1-3) amplifying an expression frame of hr3-hsp70-Cas9-sv40 from a vector pUC57-hr3-hsp70-Cas9-sv40 with the nucleotide sequence shown as SEQ ID NO. 9; then connected to AscI site of pB-Modified { IE2-Zeocin-Ser1PA } by a seamless cloning method to construct an intermediate vector pB-Modified { IE2-Zeocin-Ser1PA } { hr3-hsp70-Cas9-SV40}, and the nucleotide sequence of the intermediate vector is shown as SEQ ID NO. 11;

(1-4) amplifying U6-gRNA from a vector pUC57-U6-gRNA with the nucleotide sequence shown in SEQ ID NO.12, connecting to a vector pB-Modified { IE2-Zeocin-Ser1PA } { hr3-hsp70-Cas9-SV40} by using an enzyme digestion connection method, and constructing a eukaryotic gene knockout basic vector pB-Modified { IE2-Zeocin-Ser1PA } { U6-gRNA } { hr 3-Cas 70-Cas9-SV40}, wherein the vector is named as pB-CRISPR.

3. The construction method according to claim 1, wherein in step (3), based on CRISPR/Cas9 action rules, the targeting sites designed for realizing eukaryotic gene knockout are 23 nucleotides in total, and have the following rules:

5 '-NNNNNNNNNNNNNNNNNNNNNNNNN-NGG-3'; on the basis, a primer pair of the double gRNA vector is constructed, and has the following rule:

the forward primer is > X-F,

5-ACCGATCGATGAAGACAGAAGTGNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAA, the nucleotide sequence of which is shown in SEQ ID NO. 4;

the reverse primer is > Y-R,

5-TGATGATGATGAAGACGTAAACNNNNNNNNNNNNNNNNNNNNCACTTGTAGAGCACGAT, the nucleotide sequence of which is shown in SEQ ID NO. 5;

wherein "NNNNNNNNNNNNNNNNNNNN" in > X-F and > Y-R are targeting site X and targeting site Y, respectively.

4. The method according to claim 1, wherein in the step (5), the pB-Dul-CRISPR-XY and piggyBac transposon expression vector A3-helper (nucleotide sequence shown in SEQ ID NO. 3) are expressed in a molar ratio of 1: 1, transfecting eukaryotic cells, and screening the transfected cells by Zeocin for 2 months to obtain a cell line with two genes knocked out or functional genome fragments deleted simultaneously.

5. The method of claim 1, wherein the eukaryotic organism includes, but is not limited to, bombyx mori, drosophila, and the like.

6. A eukaryotic CRISPR-Cas double gRNA vector constructed by the method of any one of claims 1-5.

7. Use of the vector of claim 6 for the construction of a cell line for the simultaneous knock-out of two genes or the deletion of a functional gene fragment in eukaryotes.

Technical Field

The invention belongs to the technical field of eukaryotic gene knockout, and relates to a eukaryotic CRISPR-Cas9 double gRNA vector and a construction method thereof.

Background

Since the completion of human genome project, more and more model organisms and non-model organisms including mice, fruit flies, silkworms, arabidopsis thaliana, rice and the like complete whole genome sequencing, in the face of massive genome information, reading functional gene composition is an important topic of the later genome era, various genetic manipulation technologies (transgenosis, RNAi and the like) provide a basic platform for functional genome research, and among numerous genetic manipulation technologies, a gene knockout technology and a transgenic technology are two key genetic manipulation technologies for researching functional genes.

Gene editing technology is an important genetic manipulation technology developed in recent years, and four generations of genetic manipulations including meganucleases, zinc finger nucleases, transcription factor activator nucleases, CRISPR, and the like have been developed. Unlike the previous three generations of gene editing technology (relying on protein-nucleotide mutual recognition), CRISPR technology is a completely new gene editing technology based on RNA and DNA base complementary pairing. Since the invention of the CRISPR system, gene knockout has been successfully realized in many organisms including human, mouse, fruit fly, zebra fish, silkworm, Arabidopsis, tobacco, rice and the like. The efficient gene knockout cannot be separated from the efficient delivery system, the most widely applied delivery system in animals at present is a lentivirus-mediated delivery system, and the CRISPR system has been successfully delivered in mammals such as human, mice and the like. However, the lentivirus system has two significant disadvantages, one is that the lentivirus system has limited active species and is very inefficient in animals such as insects; the second is that the load bearing capacity of the lentivirus system is limited, only thousands of nucleotides, and the delivery capacity is significantly reduced with the increase of the foreign gene. In order to realize efficient gene knockout in eukaryotes, it is urgently needed to develop a system for efficiently delivering CRISPR.

The piggyBac transposon system is a type ii transposon originally found in trichoplusia ni, 2476bp in length, comprising two Inverted Terminal Repeats (ITRs) and an expression cassette encoding a transposase. The piggyBac transposable subsystem realizes transposition by adopting a shearing-sticking mode, and has high transposition efficiency. Currently, piggyBac transposable systems have been demonstrated to transpose efficiently in many species, from insects to mammals. The piggyBac transposon system has strong carrying capacity, and has been reported to carry nucleotides of more than 200kb at most, and the carrying capacity far exceeds that of a lentivirus system. Therefore, the development of the piggyBac transposon system mediated eukaryote CRISPR knockout system has wide application prospect.

On the genome, the encoded proteins only account for a small proportion, most regions are non-coding regions, and at present, researches prove that the non-coding regions of the genome have important functions, and to realize the functional deletion of the regions, the DNA sequence of the segment needs to be completely or partially deleted, so that the DNA double strand breaks at two sites on the genome are required to be simultaneously realized, and then the functional genome segment is deleted by virtue of the DNA repair channel of the cell, so that the functional deletion of the functional genome segment is realized. At present, the deletion of a DNA fragment on a genome can be realized by means of a double gRNA system, and the double gRNA system has important technical value for researching a non-coding region on a functional genome.

Disclosure of Invention

In view of this, the invention aims to provide a double gRNA vector of eukaryotic CRISPR-Cas9 and a construction method thereof.

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

1. a construction method of a double gRNA vector of eukaryotic organism CRISPR-Cas9 comprises the following specific steps:

(1) constructing a piggyBac transposon system mediated eukaryote CRISPR-Cas9 double gRNA framework vector, namely pB-CRISPR, wherein the nucleotide sequence of the vector is shown as SEQ ID NO. 1; the gene element delivery system of the vector is a piggyBac transposon system, and the gene knockout system is a CRISPR/Cas9 system;

(2) constructing a template vector for providing sgRNA scaffold and U6 promoters, namely T-DGP-7, wherein the nucleotide sequence of the template vector is shown as SEQ ID NO. 2;

(3) designing a targeting site, constructing a primer pair of a double gRNA vector, and then performing PCR amplification by using the primer pair with the T-DGP-7 obtained in the step (2) as a template to obtain an amplification product named as PCR-DGP 7-XY;

(4) digesting the pB-CRISPR obtained in the step (1) by using an endonuclease AarI as a framework, digesting the PCR-DGP7-XY obtained in the step (3) by using BbsI as a fragment, and connecting the framework and the fragment to form a double gRNA vector which is named as pB-Dul-CRISPR-XY;

(5) and (3) mixedly transfecting the pB-Dul-CRISPR-XY obtained in the step (4) and a piggyBac transposon expression vector A3-helper with a nucleotide sequence shown as SEQ ID NO.3 to a eukaryotic cell, and screening to obtain the vector.

As one of the preferable technical proposal, the specific method of the step (1) is as follows:

(1-1) synthesizing a vector PUC57-IE2-Zeocin-Ser1PA containing a Zeocin resistance gene expression cassette, wherein the nucleotide sequence of the vector is shown as SEQ ID NO. 6;

(1-2) connecting a Zeocin resistance gene expression frame IE2-Zeocin-Ser1PA on a vector PUC57-IE2-Zeocin-Ser1PA to a piggyBac transposon basic vector piggyBacModify with a nucleotide sequence shown as SEQ ID No.7 to construct an intermediate vector pB-Modified { IE2-Zeocin-Ser1PA }, wherein the nucleotide sequence is shown as SEQ ID No. 8;

(1-3) amplifying an expression frame of hr3-hsp70-Cas9-sv40 from a vector pUC57-hr3-hsp70-Cas9-sv40 with the nucleotide sequence shown as SEQ ID NO. 9; then connected to AscI site of pB-Modified { IE2-Zeocin-Ser1PA } by a seamless cloning method to construct an intermediate vector pB-Modified { IE2-Zeocin-Ser1PA } { hr3-hsp70-Cas9-SV40}, and the nucleotide sequence of the intermediate vector is shown as SEQ ID NO. 11;

(1-4) amplifying U6-gRNA from a vector pUC57-U6-gRNA with the nucleotide sequence shown in SEQ ID NO.12, connecting to a vector pB-Modified { IE2-Zeocin-Ser1PA } { hr3-hsp70-Cas9-SV40} by using an enzyme digestion connection method, and constructing a eukaryotic gene knockout basic vector pB-Modified { IE2-Zeocin-Ser1PA } { U6-gRNA } { hr 3-Cas 70-Cas9-SV40}, wherein the vector is named as pB-CRISPR.

The vector map is shown in FIG. 2.

As one of the preferable technical schemes, in the step (1-3), the vector PUC57-Hr3-Hsp70-Cas9-SV40 is obtained by replacing the promoter A4 with Hsp70 from pUC57-hA4-Cas9 (the nucleotide sequence is shown in SEQ ID NO.10, PMID: 24671069).

As one of the preferred technical solutions, in step (3), based on the CRISPR/Cas9 law of action, a targeting site for realizing eukaryotic gene knockout is designed, with 23 nucleotides in total, and the following rules are provided:

5 '-NNNNNNNNNNNNNNNNNNNNNNNNN-NGG-3'; on the basis, a primer pair of the double gRNA vector is constructed, and has the following rule:

the forward primer is > X-F,

5-ACCGATCGATGAAGACAGAAGTGNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAA, the nucleotide sequence of which is shown in SEQ ID NO. 4;

the reverse primer is > Y-R,

5-TGATGATGATGAAGACGTAAACNNNNNNNNNNNNNNNNNNNNCACTTGTAGAGCACGAT, the nucleotide sequence of which is shown in SEQ ID NO. 5;

wherein "NNNNNNNNNNNNNNNNNNNN" in > X-F and > Y-R are targeting site X and targeting site Y, respectively.

As one of the preferred technical schemes, in the step (5), pB-Dul-CRISPR-XY and piggyBactransposon expression vector A3-helper (nucleotide sequence is shown as SEQ ID NO. 3) are expressed according to a molar ratio of 1: 1, transfecting eukaryotic cells, and screening the transfected cells by Zeocin for 2 months to obtain a cell line with two genes knocked out or functional genome fragments deleted simultaneously.

As one of the preferred technical schemes, the eukaryote includes but is not limited to silkworm, fruit fly and the like.

2. The eukaryotic CRISPR-Cas double gRNA vector is constructed by the method.

3. The vector is applied to the construction of a cell line for realizing the simultaneous knockout of two genes or the deletion of functional genome segments of eukaryotes.

As one of the preferred technical schemes, the functional genome segment includes but is not limited to coding gene, non-coding RNA, genome functional element and the like.

The invention has the beneficial effects that:

the invention discloses a construction method of a piggyBac transposon system mediated eukaryote CRISPR-Cas9 double gRNA vector. The method is characterized in that by means of the ultra-large carrying capacity and wide species effectiveness of the piggyBac transposon system, a Cas9 expression frame and a double gRNA expression frame are on the same vector, the double gRNA can be efficiently and conveniently played in numerous eukaryotes, and two genes can be simultaneously knocked out or functional genome fragments (coding genes, non-coding RNAs, genome functional elements and the like) can be deleted. At present, a lentivirus system is widely applied to deliver CRISPR whole genome editing libraries to eukaryotic cells, but the lentivirus system has low efficiency in species other than mammals, and the bearing capacity is only a few kb, so that the application of the lentivirus system is limited. Because the piggyBac transposon is realized in a 'shearing-sticking' mode to realize transposition, the copy number of the exogenous gene carried by the piggyBac transposon system integrated into a host cell can be controlled by controlling the transposon concentration and the like. Compared with the lentivirus-mediated CRISPR-Cas9 single-gene knockout system widely applied at present, the method has remarkable advantages.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 is a construction process of a piggyBac transposon system mediated eukaryotic CRISPR-Cas9 double gRNA vector.

FIG. 2 is a vector pB-CRISPR map comprising: piggyBacL/piggyBacR, piggyBac swivel arm; IE2, IE2 promoter; zeocin, Zeocin resistance gene; ser1PA, bombyx mori sericin 1(Ser1) gene polyA; u6, U6 promoter; gRNA, sgRNA scaffold; hr3-hsp70, the Hr3 enhancer and the hsp70 promoter; spCas9, spCas9 protein; SV40PA, SV40 polyA.

FIG. 3 is a map of vector T-DGP7, comprising: u6, U6 promoter; gRNA, sgRNA scaffold.

FIG. 4 is a schematic diagram of a double gRNA vector system for deleting specific DNA fragments of silkworm cells, wherein the targeting sites of the double gRNAs on ME1 and ME2 are both outside a green fluorescent protein (EGFP) coding sequence, and the deleted fragments are 2791bp and 1448bp respectively; the targeting sites of ME3 and ME4 were both on the green fluorescent protein (EGFP) coding sequence, and the deletion fragments were 309bp and 61bp, respectively.

Fig. 5 is a histogram of the efficiency of deleting specific DNA fragments of silkworm cells by the dual gRNA vector system, wherein the targeting sites of the dual gRNA to ME1 and ME2 are both outside the green fluorescent protein (EGFP) coding sequence, which indicates that the efficiency of deleting specific DNA fragments of silkworm cells by the dual gRNA vector system is about 20% to 25%; the targeting sites of ME3 and ME4 are both on the coding sequence of green fluorescent protein (EGFP), which can indicate that the efficiency of the double gRNA vector system for knocking out the specific DNA of silkworm cells is over 90%.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

All the following specific experimental methods, which are not indicated, are carried out according to accepted experimental methods and conditions, for example, according to the instructions provided by the manufacturers of reagents and consumables, or according to the classic laboratory book "molecular cloning guidelines" (third edition, J. SammBruke et al).