阅读说明：本技术 一种真核生物CRISPR/Cas9全基因组编辑细胞文库及构建方法 (Eukaryotic organism CRISPR/Cas9 whole genome editing cell library and construction method ) 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全基因组编辑细胞文库及构建方法。本发明利用piggyBac转座子系统递送法构建真核生物CRISPR/Cas9全基因组编辑细胞文库,先将CRISPR/Cas9全基因组编辑系统构建到piggyBac转座子载体上,然后稳定转染细胞,筛选获得稳定表达CRISPR/Cas9文库的细胞系,再进行功能基因组筛选。本发明将Cas9蛋白表达框和sgRNA表达框整合到了同一个表达载体上,能够在从低等动物如昆虫到高等动物如哺乳动物等大多数真核生物中高效构建全基因组敲除细胞库。能够高效的在各种真核生物中实现全基因组编辑,可以高通量的在全基因组水平筛选功能基因。(The invention relates to a eukaryotic organism CRISPR/Cas9 whole genome editing cell library and a construction method thereof. The invention utilizes a piggyBac transposon system delivery method to construct a eukaryotic CRISPR/Cas9 whole genome editing cell library, firstly constructs a CRISPR/Cas9 whole genome editing system on a piggyBac transposon vector, then stably transfects cells, screens to obtain a cell line for stably expressing the CRISPR/Cas9 library, and then screens functional genomes. According to the invention, a Cas9 protein expression frame and a sgRNA expression frame are integrated on the same expression vector, so that a whole genome knockout cell bank can be efficiently constructed in most of eukaryotes from lower animals such as insects to higher animals such as mammals. Can efficiently realize whole genome editing in various eukaryotes, and can screen functional genes at the whole genome level in high flux.)

1. A method for constructing a eukaryotic CRISPR/Cas9 whole genome knockout cell library is characterized by comprising the following steps:

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

(2) constructing a gene knockout vector library pB-CRISPR-library: firstly, synthesizing a single-stranded oligonucleotide library containing a targeting site, and then cloning the single-stranded oligonucleotide library onto the vector pB-CRISPR obtained in the step (1) to successfully construct;

(3) mixing the vector library pB-CRISPR-library constructed in the step (2) with a piggyBac transposon expression vector A3-helper with a nucleotide sequence shown as SEQ ID NO.2 according to a molar ratio of 1: 1, transfecting eukaryotic host cells, and screening the transfected cells by Zeocin for 2 months to obtain the cell library.

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

(1-1) synthesizing a vector PUC57-IE2-Zeocin-Ser1PA containing an expression cassette for a Zeocin resistance gene;

(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 to construct an intermediate vector pB-Modified { IE2-Zeocin-Ser1PA };

(1-3) amplifying an expression frame of hr3-hsp70-Cas9-SV40 from a vector pUC57-hr3-hsp70-Cas9-SV40, and then connecting the expression frame to pB-Modified { IE2-Zeocin-Ser1PA } by a seamless cloning method to construct an intermediate vector pB-Modified { IE2-Zeocin-Ser1PA } { hr3-hsp70-Cas9-SV40 };

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

wherein, the nucleotide sequence of piggyBacModify is shown in SEQ ID NO. 3;

the vector PUC57-IE2-Zeocin-Ser1PA has a nucleotide sequence shown in SEQ ID NO. 4;

the nucleotide sequence of the intermediate vector pB-Modified { IE2-Zeocin-Ser1PA } is shown in SEQ ID NO. 5;

the vector pUC57-hr3-hsp70-Cas9-sv40, the nucleotide sequence is shown in SEQ ID NO. 6;

the intermediate vector pB-Modified { IE2-Zeocin-Ser1PA } { hr3-hsp70-Cas9-SV40}, and the nucleotide sequence is shown as SEQ ID NO. 8;

the nucleotide sequence of the vector pUC57-U6-gRNA is shown in SEQ ID NO. 9.

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

(2-1) designing targeting sites, designing about 6 targeting sites for each gene, and synthesizing a single-stranded oligonucleotide library containing the targeting sites by means of a DNA chip;

and (2-2) cloning the single-stranded oligonucleotide library obtained in the step (2-1) to the vector pB-CRISPR obtained in the step (1) to construct a gene knockout vector library pB-CRISPR-library.

4. The construction method according to claim 3, wherein in step (2-1), as a further preferred technical solution, in step (2-1), the knockout sites of all the genes encoding proteins are designed at the eukaryotic genome level according to the SpCas9 action rule, and the targeting sites thereof have the following rules:

5 '-NNNNNNNNNNNNNNNNNNNNN-NGG-3', the designed sgRNA sequence is consistent with the sequence of the target site on the genome, and the following rules are provided: 5 '-G-nnnnnnnnnnnnnnnnnnnnnnnnn-3'; designing the target-targeting sites of the genes of all eukaryotic coding proteins according to the rule.

5. The construction method according to claim 3, wherein in the step (2-2), the single-stranded oligonucleotide library synthesized in the step (2-1) is cloned to the pB-CRISPR vector obtained in the step (1) by using a bypass PCR technology and an enzyme digestion ligation technology to construct a gene knockout vector library pB-CRISPR-library.

6. The method for constructing a recombinant plasmid of claim 1, wherein in step (3), the transfection method is a lipofection method, the transfected cells are firstly cultured for one month under 27 ℃ condition by using a complete culture medium, the complete culture medium is Grace's culture medium comprising fetal bovine serum with the volume concentration of 10% and penicillin-streptomycin, until the cell state returns to normal, and then the complete culture medium containing Zeocin is used for continuous screening for 2 months until the genome of all the cells stably integrates the piggyBac transposon mediated CRISPR/Cas9 system, so that the cell library can be obtained.

7. The method according to claim 1, wherein in step (3), the eukaryotic host cells include BmE cells and S2 cells.

8. A eukaryotic CRISPR/Cas9 whole genome knockout cell library constructed using the method of any one of claims 1-7.

9. A method for the whole genome-wide high-throughput research of unknown functional genes of a genome by using the cell library of claim 8 comprises the following steps: based on the cell library, after screening is carried out on the library, genome DNA of cells obtained through screening is extracted, a primer pair is designed, sgRNA fragments of the cell library obtained through screening are obtained in a PCR amplification mode, the change of sgRNA abundance is analyzed through high-throughput sequencing, and then the biological function of genes corresponding to the sgRNAs is analyzed.

Technical Field

The invention belongs to the technical field of gene editing and high-throughput sequencing, and relates to a eukaryotic CRISPR/Cas9 whole genome editing cell library and a construction method thereof.

Background

The gene editing technology is an important technical means for researching functional genome, and four generations of gene editing technology has been developed at present: meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, CRISPRs, and the like. CRISPR systems are widely used because of their high efficiency and low cost. At present, the CRISPR/Cas9 system successfully realizes gene knockout in human, mouse, zebra fish, Arabidopsis, rice, fruit fly, silkworm and other species.

In addition to gene knockdown, the CRISPR/Cas9 system has also been developed for applications such as transcriptional regulation, epigenetic modification, single base editing, and the like. In addition to editing a single site, the CRISPR/Cas9 system has also been developed as a high-throughput gene knockout system for high-throughput screening of functional genes. Among high-throughput knockout gene systems, the vector library delivery systems widely used at present are lentivirus systems and site-specific nuclease systems, but these systems have some disadvantages, which limit the application of high-throughput knockout screening. For example, the load bearing capacity of a lentiviral vector is limited, the size of an exogenous gene which can be carried is generally less than 8kb, and the titer is reduced along with the increase of the length of an inserted fragment, so that a lentiviral-mediated CRISPR/Cas9 gene knockout library generally integrates a Cas9 expression cassette and a sgRNA expression cassette on the genome of a host cell in two times, and the period, cost and difficulty for constructing the CRISPR/Cas9 gene knockout library are increased; the CRISPR/Cas9 knockout library can not be widely applied to various types of eukaryotes, mainly applied to mammals, and the lentivirus system can not deliver the CRISPR/Cas9 knockout library in insects and other species.

piggyBac transposons, which are the second type of transposons originally found in lepidopteran insects and are eukaryotes, are transposed in a "cut-and-stick" fashion, easily controlling the copy number of foreign genes integrated into the host genome. The host range of the piggyBac system is wide, the high-efficiency transposition can be realized by lower animals such as insects (PMID:10625397) to higher animals such as mammals (PMID:28062688), and meanwhile, the exogenous gene fragment carried by the piggyBac transposon is extremely large and can reach 207kb (PMID: 23519027). By applying the piggyBac system, a Cas9 protein expression frame and a sgRNA expression frame can be constructed on the same piggyBac transposition carrier, and the CRISPR system can be integrated on a host cell genome only by one-step transfection, so that the experimental period is greatly shortened, and the experimental cost and the operation difficulty are reduced. Since the piggyBac transposon can carry a very large fragment of an exogenous gene, the piggyBac transposon vector can be used to integrate the CRISPR system and other more functional genetic elements into the host cell genome simultaneously. Since the piggyBac system can realize transposition in a large number of eukaryotes including insects and mammals, the piggyBac transposon vector can be used for integrating the CRISPR/Cas9 knockout library into more eukaryotes, thereby providing a powerful technical means for the research of functional genes of eukaryotes. The piggyBac system is applied to deliver the CRISPR gene knockout library, and has wide development and application prospects.

Disclosure of Invention

In view of the above, the present invention aims to provide a eukaryotic CRISPR/Cas9 whole genome editing cell library and a construction method thereof.

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

1. a method for constructing a eukaryotic CRISPR/Cas9 whole genome knockout cell library comprises the following steps:

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

(2) constructing a gene knockout vector library pB-CRISPR-library: firstly, synthesizing a single-stranded oligonucleotide library containing a targeting site, and then cloning the single-stranded oligonucleotide library onto the vector pB-CRISPR obtained in the step (1) to successfully construct;

(3) mixing the vector library pB-CRISPR-library constructed in the step (2) with a piggyBac transposon expression vector A3-helper with a nucleotide sequence shown as SEQ ID NO.2 according to a molar ratio of 1: 1, transfecting eukaryotic host cells, and screening the transfected cells by Zeocin for 2 months to obtain the cell library.

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 an expression cassette for a Zeocin resistance gene;

(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 to construct an intermediate vector pB-Modified { IE2-Zeocin-Ser1PA };

(1-3) amplifying an expression frame of hr3-hsp70-Cas9-SV40 from a vector pUC57-hr3-hsp70-Cas9-SV40, and then connecting the expression frame to pB-Modified { IE2-Zeocin-Ser1PA } by a seamless cloning method to construct an intermediate vector pB-Modified { IE2-Zeocin-Ser1PA } { hr3-hsp70-Cas9-SV40 };

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

wherein, the nucleotide sequence of piggyBacModify is shown in SEQ ID NO. 3;

the vector PUC57-IE2-Zeocin-Ser1PA has a nucleotide sequence shown in SEQ ID NO. 4;

the nucleotide sequence of the intermediate vector pB-Modified { IE2-Zeocin-Ser1PA } is shown in SEQ ID NO. 5;

the vector pUC57-hr3-hsp70-Cas9-sv40, the nucleotide sequence is shown in SEQ ID NO. 6;

the vector PUC57-Hr3-Hsp70-Cas9-SV40 is obtained by replacing a promoter A4 with Hsp70 by pUC57-hA4-Cas9(PMID:24671069, the nucleotide sequence is shown as SEQ ID NO. 7);

the intermediate vector pB-Modified { IE2-Zeocin-Ser1PA } { hr3-hsp70-Cas9-SV40}, and the nucleotide sequence is shown as SEQ ID NO. 8;

the nucleotide sequence of the vector pUC57-U6-gRNA is shown in SEQ ID NO. 9.

The vector map is shown in FIG. 1.

As one of the preferable technical proposal, the specific method of the step (2) is as follows:

(2-1) designing targeting sites, designing about 6 targeting sites for each gene, and synthesizing a single-stranded oligonucleotide library containing the targeting sites by means of a DNA chip;

and (2-2) cloning the single-stranded oligonucleotide library obtained in the step (2-1) to the vector pB-CRISPR obtained in the step (1) to construct a gene knockout vector library pB-CRISPR-library.

As a further preferred technical scheme, in the step (2-1), according to the action rule of SpCas9, knockout sites of all genes encoding proteins are designed at the whole genome level of eukaryotes, and the targeting sites have the following rules: 5 '-NNNNNNNNNNNNNNNNNNNNN-NGG-3', the designed sgRNA sequence is consistent with the sequence of the target site on the genome, and the following rules are provided: 5 '-G-nnnnnnnnnnnnnnnnnnnnnnnnn-3'; designing the target-targeting sites of the genes of all eukaryotic coding proteins according to the rule.

In the step (2-2), a single-stranded oligonucleotide library synthesized in the step (2-1) is cloned to the vector pB-CRISPR obtained in the step (1) by using a bypass PCR technology and an enzyme digestion ligation technology, and a gene knockout vector library pB-CRISPR-library is constructed.

As one of the preferable technical schemes, in the step (3), the transfection method is a lipofection method, the transfected cells are firstly cultured for one month under 27 ℃ condition by using a complete culture medium, the complete culture medium is Grace's culture medium (semer femtolier) comprising Fetal Bovine Serum (FBS) with a volume concentration of 10% and Penicillin-Streptomycin (penillilin-Streptomycin, 20 ten thousand units/liter, semperl) until the cell status is normal, and then the complete culture medium containing Zeocin is continuously screened for 2 months, the Zeocin is purchased from the semperl and has a working concentration of 200 μ g/ml until the CRISPR/Cas9 system of piggyBac-mediated transposon is stably integrated into the genome of all the cells, so that the cell library can be obtained.

As one of the preferable technical schemes, in the step (3), the eukaryotic host cell includes but is not limited to BmE cell (silkworm embryonic cell line), S2 cell (drosophila embryonic cell) and the like.

2. The eukaryotic CRISPR/Cas9 whole genome knockout cell library constructed by the method.

3. The method for realizing the high-throughput research of unknown functional genes of the genome in the whole genome range by utilizing the cell library comprises the following specific steps: based on the cell library, certain screening is performed on the library, such as virus infection, drug addition, extreme temperature stimulation and the like, then genomic DNA of screened cells is extracted, a primer pair is designed, sgRNA fragments of the screened cell library are obtained in a PCR amplification mode, the abundance change of the sgRNA is analyzed through high-throughput sequencing, and further the biological function of the gene corresponding to the sgRNA is analyzed.

As one of the preferable technical solutions, the primer pair includes:

the forward primer is > gD-F, 5-NNNNNNNNNNNNTAAATCACGCTTTCAATA, N represents a base A, T, G or C, and is shown as SEQ ID NO. 10;

the reverse primer is > gD-R, 5-NNNNNNNNNNNNCGACTCGGTGCCACTTT, and N represents a base A, T, G or C, as shown in SEQ ID NO. 11.

The invention has the beneficial effects that:

the invention utilizes a piggyBac transposon system delivery method to construct a eukaryotic CRISPR/Cas9 whole genome editing cell library, firstly constructs a CRISPR/Cas9 whole genome editing system (a Cas9 protein expression frame and a sgRNA expression frame library) on a piggyBac transposon vector, then stably transfects cells, screens to obtain a cell line for stably expressing the CRISPR/Cas9 library, and then screens functional genomes.

The currently commonly used eukaryotic organism CRISPR/Cas9 whole genome editing cell library is mainly delivered by a lentivirus system, but the lentivirus system has high efficiency mainly in mammals and extremely low efficiency in non-mammals, and the size of an exogenous gene carried by the lentivirus system is limited, generally less than 8kb, and the virus titer is gradually reduced along with the increase of an exogenous gene fragment.

According to the invention, the Cas9 protein expression frame and the sgRNA expression frame are integrated on the same expression vector (piggyBac transposon vector), so that a whole genome knockout cell bank can be efficiently constructed in most eukaryotes. The piggyBac transposon is transposed according to a 'shearing-sticking' mode, can conveniently control the copy number of an exogenous gene integrated into a host cell, and has important significance for executing whole genome editing. Can efficiently realize whole genome editing in various eukaryotes, and can screen functional genes at the whole genome level in high flux.

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 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. 2 shows that the bombyx mori CRISPR/Cas9 whole genome editing cell library is constructed by utilizing a piggyBac transposon system delivery method, and comprises the steps of designing and synthesizing sgRNA pool, constructing a bombyx mori whole genome editing vector library pB-CRISPR-lib and constructing a bombyx mori whole genome editing cell library BmEGCKLib.

Fig. 3 shows that the sgRNA correlation coefficient of the silkworm whole genome editing vector library and the cell library BmEGCKLib is 0.99, which indicates that the cell library BmEGCKLib is successfully constructed.

FIG. 4 shows that the number of genes containing the same number of sgRNAs is basically consistent when the silkworm whole genome editing vector library pB-CRISPR-Lib is compared with the cell library BmEGCKlib, which indicates that the cell library BmEGCKlib is successfully constructed.

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).