METHOD FOR INDUCING CCR5^32 DELETION BY USING CRISPR-CAS9 GENOME EDITING TECHNIQUE

Abstract

The present invention relates to a new method for successfully inducing the mutation of cell chemokine receptor CCR5 gene into CCR532 deletion gene by using the CRISPR-Cas9 genome editing technique. CCR5 is an important co-receptor for entry of Human Immunodeficiency Virus (HIV) into human host cells. CCR532 deletion is a 32-bp deletion in CCR5 coding region, which results in change and premature termination in the sequence following the 185.sup.th amino acid. Biallelic homozygous deletion of CCR532 is naturally resistant to HIV infection, i.e., the people carrying this mutation can't be infected by HIV. The present invention uses both lentiviral packaging system and the CRISPR technique to induce CCR532 deletion. Due to the characteristics of a wide range of lentivirus infection, the invention can be applied to cells such as bone marrow stem cells and CD4+ T cells and can be expected to be the therapeutic drug for HIV/AIDS infection or other diseases.

Claims

1. A method for inducing CCR532 deletion by using CRISPR-Cas9 genome editing technique which is the genome editing techniqueclustered regularly interspaced short palindromic repeats and its associated protein CRISPR-Cas9 technique, wherein the method includes the following steps: Step 1, design of a guide RNA (gRNA) For the purpose of obtaining CCR532/32 homozygous cell, a pair of gRNAs for target sequences on both sides of CCR532 is designed, wherein the left side target DNA sequence corresponding to the gRNA is selected from SEQ ID No.1 or any one sequence of SEQ ID Nos. 3-19; the right side target DNA sequence corresponding to the gRNA is selected from SEQ ID No.2 or any one sequence of SEQ ID Nos. 20-36; Step 2, constructing functional plasmid by inserting the DNA sequences corresponding to the gRNAs into CRISPR-Cas9 plasmid vector according to the designed gRNAs in Step 1, wherein the functional plasmid has the following characteristics: {circle around (1)} the plasmid carries Cas9 nuclease expression reading frame, other associated gene sequences of CRISPR, and lentiviral packaging signals; {circle around (2)} the plasmid is for the gRNAs at both sides of CCR532 locus, as described in the Step 1; Step 3, preparation of a lentiviral particle encapsulated with CRISPR-Cas9 plasmid: co-transfecting the CRISPR-Cas9 plasmid constructed in the Step 2 and the packaging plasmids PMD2.G and psPAX2 into HEK293T cells, and collecting cell supernatant after a period of time, wherein the supernatant contains the needed lentiviral particles; Step 4, infecting target cells with the lentiviral particles obtained in the Step 3 to obtain CCR532/32 homozygous deletion cells which are used as a new treatment for HIV/AIDS.

2. The method according to claim 1, wherein the DNA sequences corresponding to gRNAs contain at least DNA sequences 5 gactgta 3 and 5 taatgtc 3 for the CCR532 deletion purpose, wherein a pair of gRNAs is selected from any sequence of the left side target DNA sequences (SEQ ID No.1 and SEQ ID Nos. 3-19) and any sequence of the right side target DNA sequences (SEQ ID No.2 and SEQ ID Nos. 20-36), wherein a pair of gRNAs is selected as a combination of SEQ ID No.1 and any sequence from SEQ ID Nos. 20-36 or SEQ ID No.2; a pair of gRNAs is selected as a combination of SEQ ID No.3 and any sequence from SEQ ID Nos. 20-36 or SEQ ID No.2; and so on, a pair of gRNAs is selected as a combination of SEQ ID No.19 and any sequence from SEQ ID Nos. 20-36 or SEQ ID No.2; a pair of gRNAs is selected as a combination of SEQ ID No.2 and any sequence from SEQ ID Nos. 3-19 or SEQ ID No.1; a pair of gRNAs can be selected as a combination of SEQ ID No.20 and any sequence from SEQ ID Nos. 3-19 or SEQ ID No.1; and so on, a pair of gRNAs is selected as a combination of SEQ ID No.36 and any sequence from SEQ ID Nos. 3-19 or SEQ ID No.1.

3. The method according to claim 1, wherein the plasmid carries Cas9 nuclease expression reading frame in Step 2 carries Cas9 nickase which is Cas9 mutant or other Cas9 mutants or nuclease Fok I instead of Cas9 nuclease.

4. The method according to claim 1, wherein the plasmid vector is lentivirus vector or adenovirus vector or any other plasmid vector with CRISPR-Cas9.

5. The method according to claim 1, wherein the method in Step 3 is lentivirus infection or plasmid direct transfection.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 is a diagram illustrating the CCR5 protein marked with 7 transmembrane regions.

[0029] FIG. 2 is a diagram illustrating CCR5 gene. The block is CCR532 deletion gene and the arrowheads are Cas9 cleavage sites.

[0030] FIG. 3 is the lenti-CRISPR-v2 vector map used in experiment. The marked sites are restriction sites.

[0031] FIG. 4 is a flow chart of the entire process of the experiment.

[0032] FIG. 5 is representative microscope pictures illustrating the result of human Lymphocytic cell line Jurkat infected with lentivirus vector carrying green fluorescence protein (GFP); FIG. 5A is the picture under light microscope (magnification of 1010), FIG. 5B is the picture under fluorescence microscope (the same vision, magnification of 1010), the light white is the GFP positive cells, and the black is the GFP negative cells.

[0033] FIG. 6 is an agarose gel electrophoresis picture illustrating the efficiency of CRISPR-Cas9 system in CCR5 gene editing of lymphocyte cell line Jurkat detected by using T7 endonuclease (T7E1) digestion method.

[0034] FIG. 7 is the CRISPR-Cas9 cleavage on CCR5 gene in different monoclonal cell groups detected by using T7E1 digestion method.

[0035] FIG. 8 shows the sequencing maps at CCR532 locus of wild-type cells and the obtained CCR532/32 monoclonal cells.

[0036] FIG. 9 is a diagram illustrating sequence alignments of two obtained CCR532/32 monoclonal cells and wild-type CCR5 cells.

DETAILED DESCRIPTION

Examples

Example 1: Reconstruction of Plasmid Carrying CRISPR-Cas9 System and Preparation of Lentiviral Particles Encapsulated with the Reconstructed Plasmid

[0037] 1. Selection and Design of gRNA

[0038] (1) According to the object of experiment, a pair of gRNAs should be designed to edit CCR5 gene at the same time. However, due to the restriction of the CCR5 gene and the restriction of the PAM locus, the selection of the gRNA is limited. For the purpose of obtaining CCR532/32 homozygous cells, a pair of gRNAs is designed for target sequences on both sides of CCR532. The left side target DNA sequences corresponding to gRNA are listed as SEQ ID No.1 and SEQ ID Nos. 3-19; the right side target DNA sequences corresponding to the gRNA are also listed as SEQ ID No.2 and SEQ ID Nos. 20-36. A pair of gRNAs is selected from any sequence of the left side target DNA sequences (SEQ ID No.1 and SEQ ID Nos. 3-19) and any sequence of the right side target DNA sequences (SEQ ID No.2 and SEQ ID Nos. 20-36), for example, a pair of gRNAs can be selected as a combination of SEQ ID No.1 and any sequence from SEQ ID Nos. 20-36 or SEQ ID No.2; a pair of gRNAs can be selected as a combination of SEQ ID No.3 and any sequence from SEQ ID Nos. 20-36 or SEQ ID No.2; and so on, a pair of gRNAs can be selected as a combination of SEQ ID No.19 and any sequence from SEQ ID Nos. 20-36 or SEQ ID No.2; a pair of gRNAs can be selected as a combination of SEQ ID No.2 and any sequence from SEQ ID Nos. 3-19 or SEQ ID No.1; a pair of gRNAs can be selected as a combination of SEQ ID No.20 and any sequence from SEQ ID Nos. 3-19 or SEQ ID No.1; and so on, a pair of gRNAs can be selected as a combination of SEQ ID No.36 and any sequence from SEQ ID Nos. 3-19 or SEQ ID No.1.

[0039] (2) The reconstructed plasmid was obtained by adding a restriction enzyme cleavage site according to gRNA sequences, synthesizing DNAs, and ligating DNAs into BsmBI-digested lenti-CRISPR-v2 vector. The right insertion of the sequence was verified by sequencing. FIG. 4 shows the whole experimental process.

[0040] 2. Preparation of Lentiviral Particles and Identification of Infection Efficiency Thereof

[0041] (1) HEK293T cells were plated in 10 mm diameter dishes. Total cell number is 610.sup.6. The next day, two kinds of lenti-CRISPR-v2 plasmids with a pair of corresponding gRNAs obtained from the first step and lentiviral packaging plasmids PMD2.G and psPAX2 were co-transfected into cells in the microgram ratio 1 g:1 g:1 g:1 g and the cells were incubated in thermostatic incubator containing 5% CO.sub.2 at 37 C. for 16 h. Then, the resulting culture was centrifuged to remove the whole medium. The fresh DMEM complete medium (in which 10% fetal calf serum and two kinds of antibiotics Penicillin-Streptomycin were added) was added thereto for incubation. After 24 h, the cell supernatant was collected by centrifugation. This supernatant contains the lentiviral particles we needed. Aliquot the supernatant into 1.5 ml centrifuge tubes by 1 ml per tube and store in a refrigerator at 80 C. for use. Meanwhile, the pwpxld plasmid that can express the GFP protein was packaged into the lentiviral particle, so as to observe the efficiency of infection of lymphocytes by lentiviral particles.

[0042] (2) Lymphocytes were plated in 6-well plates before infection. Total cell number per well was 210.sup.5. The medium was the complete medium, which does not contain any antibiotics. The next day, the lentiviral particles encapsulated with pwpxld were added into cells. Polybrene that enables to increase the efficiency of virus infection was added at the same time and was adjusted to a concentration of 8 g/ml. The resulting culture was centrifuged at 1600 rpm at 37 C. for 1 h. After centrifugation, the medium containing virus fluid was removed and a new complete medium was added. The expression of GFP in cells was observed after incubation for 48 h. The efficiency of lentivirus infection can reach more than 80% depending on GFP expression situation in cells (see FIG. 5, the microscope photographs).

Example 2: The Result of Infecting Lymphocyte by the Lentiviral Particle Encapsulated with Reconstructed Plasmid Lenti-CRISPR-v2

[0043] 1. Lymphocytes were plated in 6-well plates before infection. Total cell number per well was 210.sup.5. The medium was the complete medium, which does not contain any antibiotics. The next day, the lentiviral particles encapsulated with lenti-CRISPR-v2 plasmid having corresponding gRNAs obtained in Example 1 and the lentiviral particle encapsulated with gRNA-free lenti-CRISPR-v2 plasmid were added into cell medium separately. Polybrene that enables to increase the efficiency of virus infection was added at the same time and was adjusted to a concentration of 8 g/ml. The resulting culture was centrifuged at 1600 rpm at 37 C. for 1 h. After centrifugation, the medium containing virus fluid was removed and a new complete medium was added. The cells were collected by centrifugation after incubation for 72 h. Half of the collected cells were transferred to a new culture flask for continuous incubation. The other half was used to extract total genomic DNA from the cells for subsequent analysis.

[0044] 2. Using the extracted genomic DNA of cells as a template, the sequence containing CCR532 locus was PCR-amplified with the forward primer SEQ ID No. 37 and the reverse primer SEQ ID No. 38. The editing efficiency of the CRISPR system was verified by T7 endonuclease (T7E1) digestion method (T7 endonuclease I, T7E1 in short, can identify and cut mismatched DNA). The experimental results were shown in FIG. 6. It can be seen from the figure that cleavage occurred in the group in which a pair of the lenti-CRISPR-v2 plasmids obtained in Example 1 was transfected, whereas cleavage did not occur in the gRNA-free empty vector group. It indicated our CRISPR-Cas9 system was working.

[0045] 3. CRISPR-Cas9 system may result in the insertion or deletion of a different number of bases at the break since it would initiate the cell self-repair pathway after DNA double-strands of the target sequence was cleaved by the CRISPR-Cas9 system. In order to purify cell genotype and get monoclonal cells, 100 cells were first taken out and diluted to a volume of 1000 l after counting. Then mixed thoroughly, 10 l of the mixture was added to a 96-well plate separately. 200 l of complete culture medium was added to each well. The resulting mixture was incubated in a 5% CO.sub.2 incubator at 37 C. The next day, the cell number of each well was observed, and the wells with only one cell were selected and labeled for further culture. After two weeks, the monoclonal cells grown from a single cell were transferred to a 24-well plate for further culture. Until the plate was covered with cells, the cells were transferred to a 6-well plate for further culture.

[0046] 4. The monoclonal cells in the 6-well plate were collected as described above. Half of the cells was used for further culture and the other half was used for extracting cell genome. A CCR5 gene sequence was also amplified using the primers described above. Then the amplification products were preliminarily identified whether the CCR5 gene was mutated in these monoclonal cells by digesting with T7E1. The experimental results were shown in FIG. 7. It can be seen that cleavage occurred in the five monoclonal cell groups (FIG. 7), which indicated a CRISPR-Cas9-mediated mutation. In the next step, we would prove whether a CCR532 deletion may occur by sequencing.

[0047] 5. Five different monoclonal cells were selected for further analyzing. First, 1 l of the PCR product of each monoclonal cell was taken out and ligated into the TA cloning vector in order to obtain 20 l of ligation product, respectively. Then, 5 l of the ligation product was taken out and transformed into E. coli competent cell DH5a. The single cloning was picked from plate and identified by sequencing. The result of sequencing indicated that among them, CCR532/32 homozygous deletion happened in 3 monoclonal cells, and another two monoclonal cells had insertion or deletion of different numbers of bases happened at CCR532 locus. Partial sequencing chromatograms were shown in FIG. 8. Partial sequence alignments were shown in FIG. 9.

[0048] The present invention utilizes the lentiviral particle to encapsulate our designed CRISPR-Cas9 system to infect lymphocytes, and successfully induces CCR532/32 homozygous deletion. Due to a wide range of lentivirus infection, this system can also be used to infect various types of cells such as neuronal cells, hepatocytes, cardiomyocytes, tumor cells, endothelial cells, stem cells and lymphocytes.