CN106967716A - Double gRNA, double gRNA libraries, double gRNA vector libraries and its preparation method and application - Google Patents

Abstract

The invention discloses a kind of couple of gRNA, double gRNA libraries, double gRNA vector libraries and its preparation method and application.This couple of gRNA is sequentially connected with and formed by the first promoter, gRNA a, crRNA stent sequence, the second promoter and gRNA b.This pair of gRNA library includes the multiple couples of gRNA for target gene.This pair of gRNA vector library is formed by connecting by this pair of gRNA library with carrier.In double gRNA of the present invention, gRNA a and gRNA b control it independently to express by different promoters respectively, and respectively with two target site complementary pairings on target gene, this this couple of gRNA to recruit Cas9 nucleases to shear target gene in two sgRNA action sites, effectively improve the knockout efficiency of target gene.

Description

Double gRNA, double gRNA library, double gRNA carrier library and preparation method and application thereof

technical field

The invention belongs to the field of genetic engineering, and in particular relates to a double gRNA, a double gRNA library, a double gRNA carrier library and a preparation method and application thereof.

Background technique

It is well known that the transcription of protein-coding genes only accounts for about 2% of the whole genome transcription, and the rest of the transcription is the transcription of non-coding RNA such as microRNA and lncRNA. Among them, lncRNA is defined as a group of non-coding RNAs with a molecular weight greater than 200 nucleotides in length. More and more studies have shown that lncRNA may serve as the main gene regulator in various mechanisms, and the disorder of lncRNA expression is usually associated with a variety of Human diseases, such as cancer, are closely related.

So far, more than 50,000 lncRNAs have been discovered, far more than the number of protein-coding genes, providing further evidence of the complexity of our human genome. lncRNAs are a rich source of new cancer biomarkers or therapeutic targets, and the biological functions of most lncRNAs are still unknown, so it is very important to conduct functional research on lncRNAs.

At present, the most commonly used gene functional research platform is to knock out target genes in cells through RNA interference (RNAi), specifically knock out or turn off the expression of specific genes, and then detect the interference results, and understand the relevant genes by analyzing the interference results. Function.

Unfortunately, RNAi primarily functions functionally in the cytoplasm, where the RISC complex is also located. However, many lncRNAs are located in the nucleus, which greatly increases the difficulty of knocking out lncRNAs by RNA interference. At the same time, RNAi has the following disadvantages: (1) It is difficult to carry out "reversion experiment", because the exogenous counterpart is also constrained by the RNAi reagent; (2) The knockout efficiency is not high, and high-level false negative expression is very easy to occur , especially in shRNA vectors. Therefore, it is of great significance to find new methods for gene functional research.

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) is a newly emerged technology for editing targeted genes by RNA-guided Cas9 nuclease. CRISPR/Cas9 is an adaptive immune defense mechanism evolved by bacteria and archaea in response to the constant attack of viruses and plasmids. In this system, crRNA (CRISPR-derived RNA) combines with tracrRNA (trans-activating RNA) through base pairing Forming double-stranded RNA, this tracrRNA/crRNA binary complex guides the Cas9 protein to cut double-stranded DNA at the target site of the crRNA guide sequence. During the genome editing process, tracrRNA and crRNA can be fused into one RNA (single guide RNA, referred to as sgRNA) for expression, and sgRNA can also play the role of targeted cleavage. The sequence of sgRNA contains a fixed sequence (i.e. crRNA scaffold sequence) and a target DNA recognition sequence, wherein the target DNA recognition sequence can perform base pairing with one of the two strands of the target gene, and the fixed sequence is used to recruit Cas9 nuclease.

Given the high efficiency of the CRISPR/Cas9 system, knockout of protein-coding genes is relatively easy, because a small base deletion or insertion can destroy the open reading frame, resulting in non-expression of functional gene products. However, the situation of non-coding genes, especially lncRNAs, is very different. A small base deletion or insertion does not necessarily lead to the loss of lncRNA function. How to further improve the CRISPR/Cas9 gene editing technology to realize The efficient knockout of all genes or genetic elements is a problem that needs to be considered and solved in current research.

Contents of the invention

The present invention provides a double gRNA, which has a higher knockout efficiency than sgRNA molecules.

A double gRNA (dual gRNA) is formed by sequentially linking the first promoter, gRNA-a, crRNA scaffold sequence, the second promoter and gRNA-b. A double gRNA molecule targets the same gene or genetic element, where gRNA-a and gRNA-b are independently expressed by different promoters and are complementary to two target sites on the same target gene . Therefore, the double gRNA can recruit Cas9 nuclease to cut the target gene at the two sgRNA action sites, greatly improving the knockout efficiency of the target gene.

The present invention also provides a double gRNA library, the double gRNA library includes a plurality of double gRNA for the target gene, the sequence structure of the double gRNA is: the first promoter-(gRNA-na)-crRNA scaffold sequence-the second Second promoter-(gRNA-nb);

Wherein, n represents the number of double gRNAs, and n is an integer ≥ 1.

The double gRNA in the same double gRNA library can be designed for only one target gene, or it can be a combination of several double gRNA sets designed for multiple target genes, and one double gRNA set is aimed at one target gene.

In the present invention, the first promoter and the second promoter are different polymerase III promoters, such as H1 promoter or U6 promoter.

The present invention also provides a double gRNA carrier library, which is formed by linking the double gRNA library with a carrier. The present invention has no special requirements on the carrier, as long as the cells can be successfully transfected so that the double gRNA can be expressed in the cells.

Preferably, the carrier carries a reporter gene or a resistance gene. Reporter genes or resistance genes are useful for picking positive clones in which the target gene has been knocked out.

Since artificial synthesis can only obtain mixed oligonucleotides with a length of about 200nt at one time, the double gRNA of the present invention [the first promoter-(gRNA-a)-crRNA scaffold sequence-the second promoter-(gRNA-b )] has a total length of more than 400nt, which is difficult to synthesize at one time.

Therefore, the present invention also provides a construction method of the double gRNA carrier library, the construction method comprising:

(1) Aiming at a dual gRNA action site, synthesize a single-stranded oligonucleotide with the sequence structure of "PCR amplified short sequence I-(gRNA-a)-spacer sequence-(gRNA-b)-PCR amplified short sequence II" Nucleotides, wherein both ends of the spacer sequence have restriction endonuclease cutting sites of type II;

Wherein, the enzyme cutting site can be BsmB I, Sap I, BSA I;

The base sequence of the PCR amplified short sequence I is (as shown in SEQ ID No.1):

5'-GTATGAGACCACTTGGATCC-3';

The base sequence of the PCR amplified short sequence II is (as shown in SEQ ID No.2):

5'-CCTTATTTTAACTTGCTATT-3';

Mix all single-stranded oligonucleotides synthesized for the same target gene in equal amounts, or mix equal amounts of single-stranded oligonucleotides synthesized for all target genes to obtain a mixed single-stranded oligonucleotide library;

The above-mentioned mixed single-stranded oligonucleotide library can be synthesized by the US customarray company through the "custom chip" method;

(2) Using the mixed single-stranded oligonucleotide library in step (1) as a template, using corresponding primers to perform PCR amplification to obtain a mixed double-stranded oligonucleotide library;

The upstream and downstream primers are respectively combined with PCR amplified short sequence I and PCR amplified short sequence II in single-stranded oligonucleotides to amplify and obtain a mixed double-stranded oligonucleotide library; not only double-stranded oligonucleotides can be obtained by PCR amplification Oligonucleotides for subsequent ligation into the vector, and can increase the number of clones of single-stranded oligonucleotides.

(3) Take the carrier linearized by class II restriction endonucleases, connect the mixed double-stranded oligonucleotide library in step (2) into the downstream of the first promoter of the carrier, and obtain the early stage library;

The class II restriction endonuclease in this step can be the same as step (1) or different, as long as the class II restriction endonuclease can cut at the enzyme cutting site described in step (1) .

The present invention has no requirements on the source of the first promoter and the second promoter, and the first promoter may be carried by the vector itself, or may be an exogenous first promoter recombined into the vector.

(4) The synthetic sequence structure is a single-stranded oligonucleotide library of "PCR amplified short sequence I-crRNA scaffold sequence-the second promoter-PCR amplified short sequence II";

Wherein, the base sequence of the crRNA scaffold sequence is (as shown in SEQ ID No.3):

5'-GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCC-

-GTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCT-3';

In this step, the nucleotide sequences of the PCR amplified short sequence I and the PCR amplified short sequence II are the same as in step (1);

(5) Using the single-stranded oligonucleotide library in step (4) as a template, using corresponding primers to perform PCR amplification to obtain a double-stranded oligonucleotide library;

(6) In the presence of class II restriction endonucleases, connect the double-stranded oligonucleotide library in step (5) into the previous library in step (3) by Gibson assembly method to obtain the double gRNA vector library.

The class II restriction endonuclease in this step can be the same as step (1) or different, as long as the class II restriction endonuclease can cut at the enzyme cutting site described in step (1) .

Class II restriction endonuclease cuts at both ends of the spacer sequence in the previous library, so that the double-stranded oligonucleotide library in step (5) can be connected to the position of the spacer sequence to obtain a double-stranded oligonucleotide with a complete double-gRNA sequence structure. gRNA vector library.

In view of the excellent targeted knockout performance of the double gRNA of the present invention on target genes, the present invention also provides the application of the double gRNA carrier library in gene knockout.

Moreover, the double gRNA carrier library of the present invention is especially suitable for knocking out lncRNA.

In this regard, the present invention provides two strategies for knocking out lncRNA using the double gRNA carrier library:

① For lncRNAs with coherent sequences, design gRNA-a and gRNA-b for both ends of the lncRNA, construct a double gRNA carrier library, and use the double gRNA carrier library to transfect or infect cells to knock out the target lncRNA in the cells;

②For the lncRNA with other genes or genetic elements inserted in the sequence, use the first exon in the lncRNA and its upstream promoter as the target, design gRNA-a and gRNA-b, construct a double gRNA vector library, and The cells are transfected or infected with the double gRNA vector library to knock out the target lncRNA in the cells.

The present invention also provides a gene knockout cell library for studying corresponding gene functions, the gene knockout cell library is obtained by transfecting or infecting the double gRNA vector library into cells stably expressing Cas9 nuclease.

After the double gRNA carrier library is transfected or infected into cells stably expressing Cas9 nuclease, it can recruit Cas9 nuclease to knock out the target gene corresponding to the double gRNA, and the cell phenotype after the target gene is knocked out changes, thereby Can be used to determine target gene function.

Compared with prior art, the beneficial effect of the present invention is:

(1) Compared with gRNA, in the double gRNA of the present invention, gRNA-a and gRNA-b are independently expressed by different promoters, and are complementary to two target sites on the target gene, which makes The double gRNA can recruit Cas9 nuclease to cut the target gene at the two gRNA action sites, and the knockout efficiency of protein-coding genes can reach 100%, while the knockout efficiency of gRNA can only reach 66.7%;

(2) Compared with siRNA and shRNA, the double gRNA of the present invention can restore the experiment and verify the function of the knockout gene.

Description of drawings

Fig. 1 is the construction flowchart of double gRNA carrier library of the present invention;

Among them, H1 and U6 represent H1 and U6 promoters respectively, Lenti-hygro represents Lenti-hygro vector, Cut with BsmB I represents cutting lenti-hygro vector with restriction endonuclease BsmB I, Hygro represents hygromycin resistance Sex gene, Scaffold indicates the scaffold, First step cloning indicates the first step of cloning, Spacer indicates the spacer sequence, gRNA1 and gRNA2 respectively indicate a sgRNA, Second step cloning indicates the second step of cloning, Lenti-dualgRNA-hygro-prelibrary indicates the previous library, Lenti -dual gRNA-hygro-library means double gRNAs carrier library; the same below;

Figure 2 is a schematic diagram of the knockout mechanism of lncRNA by double gRNA;

Among them, LncRNA represents a long non-coding RNA, gRNA-1 and gRNA-2 represent a sgRNA respectively, flanking represents a flank (sequence or region), 1 ^st exon represents the first exon, and Last exon represents the last exon , Myc means Myc gene, hCas9 means human codon-optimized Cas9 nuclease gene, NLS means nuclear localization signal, deleted means that the original LncRNA has been knocked out by double gRNA; the same below;

Figure 3 is the comparison result of gene knockout efficiency of sgRNA and double gRNA;

Among them, Single gRNA means a single sgRNA, Dual gRNA means two sgRNAs (double gRNA), EF1 means promoter, E1, E2, E3 means three exons of UCA1 gene, Marker means DNA molecular weight standard, Vector means carrier, UCA1KO (2, 4, 14, 16, 19) indicate clones in which the UCA1 gene was knocked out by Single gRNA (clone numbers are 2, 4, 14, 16, 19), and UCA1KO (3, 5, 12, 17) indicate that the UCA1 gene was knocked out Dual gRNA knockout clones (clone numbers are 3, 5, 12, 17), Genomic PCR indicates the PCR results of genomic DNA; the same below;

Figure 4 is the comparison result of the knockout efficiency of double gRNA to long lncRNA under different knockout strategies;

Among them, chr8 represents chromosome 8, 1-8 represent the eight exons on the PTV1 gene, and the 3rd to 5th exons are miR-1205, 1206, 1207 respectively, Exon 1 and Exon 8 represent the first, 8 exons, E1KO means the clone with the first exon of PTV1 gene knocked out, E1+P KO means the clone with the first exon of PTV1 gene and its upstream promoter region knocked out, RelativePVT1level(% ) represents the relative expression level (%) of PTV1 gene;

Figure 5 is a diagram of the knockout effect of the double gRNA on the AK023948 gene;

Among them, AK023948KO (#13, #28, #32) means the clones with AK023948 gene knocked out (clone numbers are #13, #28, #32), and Relative AK0level (%) means the relative expression level (%) of AK023948 gene .

detailed description

The technical solution of the present invention will be described in further detail below in conjunction with the accompanying drawings and specific embodiments.

The construction of embodiment 1 double gRNA carrier library

There are more than 50,000 lncRNAs listed in the non-coding database (www.http://noncode.org/). In principle, there is no upper limit for the construction of the double gRNA carrier library of the present invention, but considering that the current U.S. customarray company uses "custom chips" The upper limit of the number of single-stranded oligonucleotides synthesized by the method, we screened about 45,000 lncRNAs suitable for sgRNA labeling, and designed two double gRNAs for each lncRNA to constitute a total of 9,000 double gRNA KO libraries. In addition, 100 non-human gene negative control double gRNAs were also set up.

The construction method of the double gRNA KO library in this embodiment (the construction process is shown in Figure 1) includes:

(1) Preparation of mixed single-stranded oligonucleotide library

Entrust the American customarray company (http://www.customarrayinc.com/) to synthesize the corresponding single-stranded oligonucleotides through the "custom chip" method to obtain a mixed single-stranded oligonucleotide library;

(2) Preparation of mixed double-stranded oligonucleotide library

Use the mixed single-stranded oligonucleotide library as a template, and use primers for PCR amplification:

In a 1mL tube, add: 80μL of 5×PCR reaction buffer, 8μL of dNTP mixture (10mM), 2μL of Phusion polymerase (2U/μL), 10μL of H1-5.1 primer (10μM), 10μL of Scaffold-adaptor primer (10μM), Template (80ng/μL) 2μL, add deionized water to a total volume of 400μL.

Wherein, the H1-5.1 primer (as shown in SEQ ID No.4) is combined at the position of "PCR amplified short sequence I" described above, and the Scaffold-adaptor primer (as shown in SEQ ID No.5) is combined at At the position of "PCR amplified short sequence II" mentioned above. The base sequences of the two are:

H1-5.1 primer: 5'-GTATGAGACCACTTGGATCC-3';

Scaffold-adaptor primer: 5'-CCTTATTTTAACTTGCTATT-3'.

Mix the PCR reaction system evenly, centrifuge it slightly, divide it into 8 PCR tubes (50μl/tube), and carry out the amplification reaction in a PCR instrument. The reaction parameters are: 98°C for 1min, 1 cycle, 98°C for 0.5min, 42°C 1min, 72°C 0.5min, 33 cycles, 72°C 4min, 1 cycle, 4°C storage.

The PCR product was purified using the Zymo Research Purification Kit of the United States:

For every 200 μL of PCR solution, 1 mL of ligation buffer was added, transferred to a purification column, and centrifuged in a centrifuge (Eppendoff) at 15,000 rpm for 0.5 min. Discard the centrifugate, add 200μL wash buffer, centrifuge at 15,000rpm for 0.5min; add 200μL wash buffer, centrifuge at 15,000rpm for 1min, transfer the spin column to a new tube, add 40μL water to the column, centrifuge at 15,000rpm for 1min, save the eluate (that is, a mixed double-stranded oligonucleotide library) for the subsequent first step of cloning.

(3) Preparation of carrier

The lenti-hygro vector was digested with BsmBI (NEB, USA). The digestion system is: Buffer3.1 10 μL, lenti-hygro carrier 2 μg, BsmB I (10 units/μL) 2 μL, add water to 100 μL, digest at 55°C for 3 hours.

1% agarose gel, 80 constant current for 30min, separate the digested DNA (using DNA tapping recovery kit from Zymo Research, USA), cut the linear DNA band, add sol solution (Zymo Research) to a 1.5mL tube, Add 300 μL of colloidal solution per 100 μg of gelatin, at 55°C for about 5 minutes, or until the gelatin is completely dissolved. Transfer to the purification column, centrifuge (Eppendoff) at 15,000rpm for 0.5min, add 200μL wash buffer, centrifuge at 15,000rpm for 0.5min; add 200μL wash buffer, centrifuge at 15,000rpm for 1min; transfer the spin column to a new tube, add 40μL water Put it into the column, centrifuge at 15,000rpm for 1min, and save the eluate for the first step of cloning.

(4) The first step of cloning (preparation of pre-library)

Add 4 μL of PCR product (i.e. mixed double-stranded oligonucleotide library), 4 μL of lenti-hygro carrier/BsmB1, 2 μL of cold fusion (SBI) into the tube, let it stand at room temperature for 5 minutes, transfer to ice and let it stand for 10 minutes before adding 50 μL of competent cells (10G purchased from Lucigen, USA) were placed on ice for 30 min, then placed in a water bath at 42°C for 1 min, then transferred to ice and allowed to stand for 2–5 min, then added with 500 μL of LB medium, and cultured with shaking at 37°C and 250 rpm for 60 min. Take 250 μL and spread it on an ampicillin (100 μg/mL) LB plate, incubate overnight at 37°C, and count the number of clones the next day.

Depending on the size of the library, we need 5-10x coverage, that is, for 10,000 double-gRNA libraries, we need 50,000 to 100,000 clones.

Use the plasmid extraction kit of Zymo Research Company in the United States to isolate the plasmid in the transformed bacteria containing the double gRNA expression vector. The extraction steps are:

Pre-library DNA 2μg, 10x buffer 3.1 (NEB, USA) 10μL, BsmB I (10units/μL) 2μL, add water to 100μL, digest at 55°C for 3 hours.

1% agarose gel, 80 constant current for 30min, separate the digested DNA (using the U.S. Zymo Research company DNA tapping gel recovery kit), cut the linear DNA band, in a 1.5mL tube, add the sol solution ((Zymo Research) , add 300μL sol solution for every 100μg gel, 55℃ for about 5mins or until the gel is completely dissolved. Transfer to the purification column, centrifuge (Eppendoff) at 15,000rpm for 0.5min, add 200μL wash buffer, centrifuge at 15,000rpm for 0.5min; add 200μL wash buffer , Centrifuge at 15,000rpm for 1min; transfer the spin column to a new tube, add 40μL of water to the column, centrifuge at 15,000rpm for 1min, and save the eluate (plasmid DNA, that is, the previous library) for the second step of cloning.

(5) Preparation of Scaffold-U6 fragment

Add 80 μL of 5×PCR reaction buffer, scaffold-U6 upstream primer (Scaffold-U6-5.1: 5'-GTTTTAGGCTAGAAAT-3') (as shown in SEQ ID No.6) and downstream primer (Scaffold- U6-3.1: 5'-ACGGTGTTTCGTCCTTTC-3') (shown in SEQ ID No.7) 10 μL (10 μM), dNTP mixture (10 mM) 8 μL, plasmid with Scaffold-U6 (100 ng/μL) 1 μL (entrusted Synthesized by American genscript company), Phusion polymerase (2U/μL) 2 μL, add deionized water to a total volume of 400 μL. After mixing, centrifuge slightly and divide into 8 PCR tubes, each tube is 50 μL, and carry out the amplification reaction in a PCR instrument. The PCR reaction parameters are: 98°C for 1min, 1 cycle, 98°C for 0.5min, 42°C 1min, 0.5min at 72°C, 33 cycles, 4min at 72°C, 1 cycle, store at 4°C for later use.

(6) The second step of cloning

Add 4 μL of PCR product (Scaffold-U6 fragment), 4 μL of pre-digested library with BsmB I, 2 μL of cold fusion (SBI), let stand at room temperature for 5 minutes, then transfer to ice for 10 minutes; add 50 μL of competent cells (10G was purchased from American Lucigen Company), put it on ice for 30 minutes, then put it in a water bath at 42°C for 1 minute, then transferred it to ice and let it stand for 2-5 minutes, then added 500 μL of LB culture medium, cultured with shaking at 37°C and 250 rpm for 60 minutes, and took 250 μL of culture medium The solution was plated on ampicillin (100 μg/mL) LB plates, incubated overnight at 37°C, and the number of clones was counted the next day.

Depending on the size of the library, we need 5-10x coverage, that is, for 10,000 double-gRNA libraries, we need 50,000 to 100,000 clones.

Use the plasmid extraction kit of Zymo Research Company in the United States to isolate the plasmid in the transformed bacteria containing the double gRNA expression vector (ie, the double gRNA vector library), purify and store it, and use it for subsequent viral packaging.

The double gRNA vector library can be used to screen essential genes that play a significant role in gene regulation and disease progression, and can also be used to identify the functions of essential coding genes or other genetic elements.

The knockout mechanism of double gRNA to LncRNA is shown in Figure 2.

The gene knockout efficiency comparison of embodiment 3 single gRNA and double gRNA

In this example, the UCA1 gene with 3 exons is taken as an example to compare the gene knockout efficiency of sgRNA and double gRNA (as shown in Figure 3).

(1) Gene knockout efficiency of double gRNA

Using the UCA1 gene as the target gene, select the target site on the target gene (as shown in part D in Figure 3), and design UCA1-dual gRNA, the base sequences of the sense strands of the two gRNAs in the UCA1-dual gRNA are respectively:

UCA1-gRNA1 (SEQ ID No.8): 5'-GTGCATGGTGGAGAGATGAT-3';

UCA1-gRNA2 (SEQ ID No.9): 5'-TTCTGGAATGGTGAACCCAA-3';

Entrust the American genscript company to construct the expression vector containing hCas9 and UCA1-dual gRNA (each DNA content is 0.5 μg), and use the expression vector to transfect and knock out 293T cells. The knockout steps are as follows:

1) Subculture 293T cells, and maintain the cell density at about 30%, overnight at 37°C;

2) The expression vector containing hCas9 and UCA1-dual gRNA (as shown in part B in FIG. 3 ) is used in a 6-well plate to transfect cells with a transfection reagent such as DNAfectin (ABM Company, USA) or lipofectin (Thermo Fisher Company, USA);

3) Change the serum medium 6 hours after transfection;

4) The next day, separate single cells by FACS cell sorter;

5) Let a single cell grow for about 10 days in a 96-well plate;

6) Transfer clones to a 24-well plate and grow for more than 5 days;

7) Genomic DNA of UCA1 knockout clones was isolated with a kit from Viagen Biotech, USA:

a) Add 200-300 μL of DirectPCR Lysis Reagent (Cell) to the cells on a 10 cm plate, and add freshly prepared 0.2-0.4 mg/mL proteinase K (Sigma) at the same time; rotate the tube in a water bath at 55°C for 1 hour, or observe until there are no lumps; 85 Cleavage in a water bath for 45 minutes. Centrifuge for 10 seconds and store at -20°C;

b) Take 0.5 μl of the lysate as a template for PCR reaction, the reaction system includes: 1 μL of 10×PCR reaction buffer, upstream primer (UCA1-inside-5.3: 5'-CCTTAACTAATTAAC--CCACC-3') (such as SEQ ID No.10) and downstream primer (UCA1-inside-3.3: 5'-AAGAGAGTCAGCGAAGGGAG-3') (as shown in SEQ ID No.11) each 0.5μL (10μM), dNTP mixture (10mM) 0.2μL, DMSO 0.3 μL, Taq polymerase (NEB, USA) (2U/μL) 0.2 μL, add 6.8 μL of deionized water to a total volume of 10 μL;

c) Carry out DNA amplification reaction in a PCR instrument. The PCR reaction parameters are: 94°C 1min, 1 cycle, 94°C 0.5min, 55°C 0.5min, 72°C 0.5min, 33 cycles, 72°C 10min, 1 cycle Cycle, store at 4°C;

d) After the reaction, 4 μL of the reaction solution was taken and subjected to 1% agarose gel electrophoresis to detect the absence of UCA1 (as shown in part F in FIG. 3 ).

(2) Gene knockout efficiency of single gRNA

Using the UCA1 gene as the target gene, select the target site on the target gene (as shown in part C in FIG. 3 ), and design UCA1-gRNA, which is the same as the UCA1-gRNA1 above.

The expression vector containing hCas9 and UCA1-gRNA (as shown in part A in Figure 3) was constructed using the same method as in part (1) of this example, and the expression vector was used to transfect target cells, and the total cells of UCA1 knockout clones were isolated. The RNA was subjected to RT-PCR to detect the expression level of UCA1 (as shown in part E in FIG. 3 ).

The steps of RT-PCR are:

a) Extraction of RNA: Extract RNA from 293T cells with Direcozol TM RNA MIN/prep kit, add 500 μl (trl reagent) (cat.R2050-100) to each well of 24 wells and place the sample on ice;

b) Frozen samples need to be mixed with a pipette tip, and an equal amount of 95% ethanol is added to mix well;

c) Transfer the mixed solution into a special (Zymo-spin) IIC column, centrifuge at 13500rpm for 1min, and replace the spin column with a new collection tube;

d) Add 400μl Direcozol TM RNA prewash to the (Zymo-spin) IIC column, and centrifuge at 13500rpm for 1min;

e) repeat step d);

f) Add 700 μl RNA wash buffer to the (Zymo-spin) IIC column, and centrifuge at 13500 rpm for 1 min. Throw away the centrifugate and centrifuge at 13500rpm for 2min;

g) Add 25μl Dnease/RNAease free water to (Zymo-spin) IIC spin column, and centrifuge at 13500rpm for 1min;

h) Utilize nucleic acid instrument Nano drop2000C to measure RNA concentration (Thermo Scientific Company of the United States);

i) Reverse transcription: put all reagents on ice, reverse transcription system: 0.5 μg RNA, 0.5 μl random primer (randomprimer mix#s1330s), add DEPC H ₂ O to 7 μl, put the PCR instrument on ice at 70°C for 10 minutes;

j) RT- add the following to each sample: Thermo scicentiitic 5X first stand buffer (lot00168122) 2μl, dNTP (signa deoxynuclrotide mix, lot susf-45360us) 0.5μl, Rnase inhibitor (munne#Mo314l) (lot10081460) 0.25μl Revert AidRT ( revertaid.lt 00205481) reverse Tanscrripatse#epo441 0.25μl, mix well, set the reaction parameters as 25°C for 10min, 37°C for 1hour, 70°C for 10min (eppendorf orBIO-RAD T100thermal cycler) to obtain template cDNA;

k) QPCR: Add 10 μL real-time PCRMaster Mix, 0.4 μL 50×ROX reference, 0.4 μL each of the corresponding PCR forward and reverse primers (10 μM), and 2 μL template cDNA (above obtained from the reverse transcription step) and 6.8 μL of deionized water; after mixing, PCR reaction was carried out. The reaction conditions were: 95°C for 3min, the cycle conditions were 95°C for 10s and 60°C for 0.5min, 65°C for 0.05s, a total of 39 cycles, each Each sample was repeated 3 times, and the expression level after UCA1 gene knockout could be obtained through this reaction.

It can be seen from Figure 3 that in the randomly selected UCA1 knockout clones, the UCA1 knockout clones knocked out by UCA1-gRNA also contained the UCA1 gene, while the UCA1 knockout clones knocked out by UCA1-dual gRNA could not be detected UCA1 gene, indicating that double gRNA has a higher knockdown efficiency than sgRNA.

Example 3 The knockout strategy of double gRNA to long lncRNA

When the lncRNA gene is too long, some other genes such as microRNA will be inserted into it. For example, the PVT1 gene has 8 exons, and its gene interval is more than 200kb (chr8:128,902,874-129,113,499), and three microRNAs (miR-1205-1207) are located in this region (part A in Figure 4). If the same knockout strategy as in Example 3 is used to cut out all the regions exceeding 200kb, these three microRNAs and some other potential genes or genetic elements may also be deleted at the same time, which will make it impossible for us to determine It is necessary to accurately judge whether the cut phenotype is related to PVT1 or other factors.

Therefore, two knockout strategies were designed for the PVT1 gene:

(1) Using the same method as in Example 3 to design a double gRNA for cutting the first exon of the PVT1 gene (as shown in Part B in Figure 4);

(2) Using the same method as in Example 3 to design a double gRNA for cutting the first exon of the PVT1 gene and the 1.1kb region upstream of the transcription of the first exon (as shown in part C in Figure 4);

Using the same method as in Example 3 (1), the expression vectors containing hCas9 and PVT1-dual gRNA (gRNA1-gRNA2) and the expression vectors containing hCas9 and PVT1-dual gRNA (gRNA1p-gRNA2) were respectively constructed, and used respectively The two expression vectors were transfected into the target cells, and the genomic DNA of the KO clone was isolated for PCR to detect the deletion of the PVT1 gene in the KO clone (as shown in parts C and D in FIG. 4 ).

It can be seen from FIG. 4 that although the first knockout strategy knocks out the first exon of the PVT1 gene, other exons of the PVT1 gene are still expressed. However, after the second knockout strategy knocked out the first exon of PVT1 gene and the 1.1 kb region upstream of the transcription of the first exon, the expression of exon 1 and exon 8 was not detected.

This is because, similar to protein-coding genes, the vast majority of lncRNAs are transcribed by RNA polymerase II. Although a promoter may contain a very large region, several enhancer regions and basic transcriptional elements are located within 500 bp upstream of transcription. To be on the safe side, we designed sgRNA1p with transcription upstream from 1KB. For example, gRNA1p of PVT1 is located in the 1.1 kb region and gRNA2 is located at the bottom of the first exon, covering a total of up to 1.5 kb. Thus, the double gRNA containing gRNA1p-gRNA2 can complete the knockout of the entire transcription of PVT1.

In addition, the double gRNA containing gRNA1p-gRNA2 had no effect on miR-2015~miR-2017, indicating that they may have corresponding promoters.

Example 4 Application of double gRNA KO library

Although the role of long noncoding RNAs (lncRNAs) has been increasingly recognized in various physiological and pathological conditions, the potential roles of large numbers of lncRNAs in breast cancer are less understood. We investigated the expression profile of lncRNAs by RT-PCR and identified a set of lncRNAs that were differentially expressed in patient breast cancer tissues relative to normal breast tissues, respectively. Among them, AK023948 is up-regulated in breast cancer, and it is also up-regulated in breast cancer cell lines (MCF-7 and MDA-MB-231) compared to human breast epithelial cells (HMLE). In situ hybridization was further validated.

This example uses AK023948 as the target gene, selects the target site on the target gene (as shown in part A in Figure 5), and designs AK023948-dual gRNA, the base sequences of the sense strands of the two gRNAs in AK023948-dual gRNA are respectively for:

AK023948-gRNA1 (as shown in SEQ ID No.12):

5'-GAGTTTTAGTCACCTATCTA-3';

AK023948-gRNA2 (as shown in SEQ ID No.13):

5'-GGTGATCCTTGTGCACGGCC-3';

Using the same method as in Example 3 (1), construct expression vectors containing hCas9 and AK023948-dual gRNA respectively, and use the expression vectors to transfect target cells, isolate the genomic DNA of KO clones for PCR, and detect The deletion of the AK023948 gene (as shown in part B in Figure 5); while using the same method as in Example 3 (2), the total RNA of the KO clone was isolated, and RT-PCR was performed to verify the presence of the AK023948 gene in the corresponding KO clone. Missing situation (shown in part C in Figure 5).

It can be seen from Figure 5 that the AK023948 gene can be completely knocked out by using the double gRNA of this embodiment.

Claims (10)

1. a kind of couple of gRNA, it is characterised in that by the first promoter, gRNA-a, crRNA branch Frame sequence, the second promoter and gRNA-b are sequentially connected with and formed.

2. a kind of pair of gRNA library, it is characterised in that this pair of gRNA library includes being directed to target base Multiple couples of gRNA of cause, described couple of gRNA sequential structure is：First promoter-(gRNA-na)- The promoter of crRNA stent sequences-the second-(gRNA-nb)；

Wherein, n represents double gRNA number, and n is >=1 integer.

3. gRNA libraries as claimed in claim 2 double, it is characterised in that the first promoter and Second promoter is mutually different polymerase III promoter.

4. a kind of pair of gRNA vector library, it is characterised in that double as described in Claims 2 or 3 GRNA libraries are formed by connecting with carrier.

5. the construction method of double gRNA vector libraries as claimed in claim 4, it is characterised in that Including：

(1) for a double gRNA action site, composition sequence structure is " PCR expands short sequence The single stranded oligonucleotide that I- (gRNA-a)-intervening sequence-(gRNA-b)-PCR expands short sequence II " is arranged, Wherein, the two ends of intervening sequence carry the restriction enzyme site of II class restriction enzymes；

All single stranded oligonucleotide mixed in equal amounts that will be synthesized for same target gene, or, it will be directed to The single stranded oligonucleotide mixed in equal amounts of all target gene synthesis, obtains mixing single stranded oligonucleotide storehouse；

(2) using the mixing single stranded oligonucleotide storehouse in step (1) as template, corresponding primer is utilized Enter performing PCR amplification, obtain mixing double chain oligonucleotide storehouse；

(3) carrier of II classes of learning from else's experience restriction enzyme linearisation, the mixing in step (2) is double Chain oligonucleotide library is connected into the first promoter downstream of the carrier, obtains early stage library；

(4) composition sequence structure is " PCR expands short sequence I-crRNA stent sequences-the second and started Son-PCR expands short sequence II " single stranded oligonucleotide storehouse；

(5) using the single stranded oligonucleotide storehouse in step (4) as template, carried out using corresponding primer PCR is expanded, and obtains double chain oligonucleotide storehouse；

(6) in the presence of II class restriction enzymes, by Gibson construction from parts by step (5) In double chain oligonucleotide storehouse be connected into the early stage library in step (3), obtain described couple of gRNA Vector library.

6. the construction method of double gRNA vector libraries as claimed in claim 5, it is characterised in that In step (1) or step (4), the base sequence that the PCR expands short sequence I is：

5’-GTATGAGACCACTTGGATCC-3’；

The base sequence that the PCR expands short sequence II is：

5’-CCTTATTTTAACTTGCTATT-3’。

7. double applications of the gRNA vector libraries in gene knockout as claimed in claim 4.

8. application as claimed in claim 7, it is characterised in that the gene is lncRNA.

9. application as claimed in claim 8, it is characterised in that including：

1. for the lncRNA of sequence context, for the lncRNA two ends design gRNA-a and GRNA-b, builds double gRNA vector libraries, and using this pair of gRNA vector libraries transfection or feel Cell is contaminated, intracellular target lncRNA is knocked out；

2. for the lncRNA inserted with other genes or genetic elements in sequence, with the lncRNA In first extron and its upstream promoter as target spot, design gRNA-a and gRNA-b, Double gRNA vector libraries are built, and using this pair of gRNA vector libraries transfection or infection cell, it is right Intracellular target lncRNA is knocked out.

10. a kind of Knockout cells library for being used to study corresponding gene function, it is characterised in that It is as claimed in claim 4 double by the transfection into the stable cell for expressing Cas9 nucleases or infection What gRNA vector libraries were obtained.