Method for inducing ccr5^32 deletion by using crispr-cas9 genome editing technique

Patent metadata

Field	Value
Publication number	US-2018363001-A1
Application number	US-201615518252-A
Country	US
Kind code	A1
Filing date	Apr 12, 2016
Priority date	Jan 18, 2016
Publication date	Dec 20, 2018
Grant date	—

Abstract

The present invention relates to a new method for successfully inducing the mutation of cell chemokine receptor CCR5 gene into CCR5Δ32 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. CCR5Δ32 deletion is a 32-bp deletion in CCR5 coding region, which results in change and premature termination in the sequence following the 185 th amino acid. Biallelic homozygous deletion of CCR5Δ32 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 CCR5Δ32 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.

First claim

What is claimed is:

1 . A method for inducing CCR5Δ32 deletion by using CRISPR-Cas9 genome editing technique which is the genome editing technique—clustered 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 CCR5Δ32/Δ32 homozygous cell, a pair of gRNAs for target sequences on both sides of CCR5Δ32 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 CCR5Δ32 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 CCR5Δ32/Δ32 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 CCR5Δ32 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.

Assignees

• Univ Nankai

Inventors

• Wei Min
• Qi Chunxia

Classifications

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C12N15/86 Primary

  Viral vectors · CPC title

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N15/11

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title