WO2019237379A1 - Modified vector for human cnih2 gene editing, preparation method therefor and application thereof - Google Patents

Abstract

Provided are a modified vector for human CNIH2 gene editing, preparation method therefor and application thereof, relating to the technical field of genetic engineering. The provided modified vector for CNIH2 gene editing can edit a human CNIH2 gene at the cellular level by means of CRISPR/Cpf1 system, so as to obtain a CNIH2 gene-edited positive cell.

Description

Modified vector for human CNIH2 gene editing, preparation method and application thereof Technical field

 The invention relates to the technical field of genetic engineering, in particular to a modified vector for human CNIH2 gene editing, a preparation method and application thereof.

Background technique

Alzheimer's disease (AD) is the most common cause of senile dementia. This disease is common in the elderly. It is a degenerative disease of the central nervous system with progressive cognitive impairment and memory impairment. The clinical manifestations are continuous deterioration of cognitive and memory functions, progressive decline in daily living ability, and Various neuropsychiatric symptoms and behavioral disorders. Many cases occur in the elderly, with latent onset, the course is slow and irreversible, and clinically, mental damage is the main cause. Increasing evidence suggests that CNIH2 is a key mechanism for regulating synaptic plasticity in the central nervous system. CNIH2 is one of the ionic glutamate receptors, which mediates the main component of central excitatory post-synaptic currents and is indispensable in the process of synaptic transmission.

technical problem

The physiological function of CNIH2 gene depends on the regulation of factors such as the number of receptors, subunit composition, and protein phosphorylation. CNIH2 dysfunction, leading to decreased synaptic plasticity of neurons, causing AD cognitive impairment, and plays an important role in the pathogenesis of AD. Therefore, CNIH2's role in Alzheimer's disease and its use as a therapeutic target Research is indispensable, but the lack of means for targeted knockout of CNIH2 gene expression in the prior art has caused certain obstacles to the progress of related research.

Technical solutions

A first object of the present invention is to provide a modified vector for human CNIH2 gene editing.

A second object of the present invention is to provide a method for preparing a modified vector for human CNIH2 gene editing.

The present invention provides a targeted sgRNA for human CNIH2 gene editing, and its sequence is 5’-AATTTCTACTCTTGTAGATCTGTCATCTGGCACGTAAGGCCG -3 ’.

The present invention also provides a method for preparing the above-mentioned modified vector for human CNIH2 gene editing, including the following steps:

(1) annealing and complementary pairing the sense and antisense strands of the sgRNA targeting the CNIH2 gene;

(2) Digest the core vector with BamHI and EcoRI endonuclease, and recover the linearized core vector by agarose gel electrophoresis;

(3) ligating the sgRNA obtained in step (1) to a linearized core vector with T4 DNA ligase;

(4) The ligation product is transformed into competent E. coli Stbl3. After a large amount of culture, the recombinant vector is extracted and commissioned for sequencing. The correct sequencing result is the modified vector for human CNIH2 gene editing.

Further, in step (2) and step (3), the core vector is pLVX-ascpf1-puro.

Beneficial effect

The modified vector for human CNIH2 gene editing provided by the present invention has strong specificity, can edit human CNIH2 gene through the CRISPR / Cpf1 system very efficiently, and can be used in research and development of drugs related to abnormal expression of CNIH2 gene.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a vector map of pLVX-AsCpf1-puro;

Figure 2 T7 Endonuclease I test results of RGC5 cells in the control group and experimental group, where: M-Marker, 1-experimental group, 2-control group.

Embodiments of the invention

The following is a preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principles of the present invention, several improvements and retouches can be made. It is the protection scope of the present invention.

The methods used in the following examples are conventional methods unless otherwise specified. The primers and DNA sequences used were synthesized by Shanghai Shengong Company.

Examples 1 : Target people CNIH2 genetic pLVX-AsCpf1-puro-CNIH2 Preparation of the carrier

a. According to Bernd Zetsche et al. (Cpf1 Is a Single The RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System) provides crRNA design rules and gRNA sequences that target the CNIH2 gene, designing crRNAs that target the CNIH2 gene, and according to the actual situation of the pLVX-AsCpf1-puro vector, the The sticky ends of BamHI and EcoRI digestion sites were added at the 3 'and 3' ends, respectively. The forward sequence was 5'-GATCCTAATTTCTACTCTTGTAGATCTGTCATCTGGCACGTAAGGCCG-3 'and the reverse sequence was 5'-GCGGCCTTACGTGCCAGATGACAGATCTACAAGAGTAGAAATTAATTC-3'. These two sequences were synthesized. After dissolution, 5 μL of each was mixed, heated at 95 ° C for 5 minutes, and then naturally cooled to room temperature to form a double-stranded DNA with sticky ends of BamHI and EcoRI.

b. Digest pLVX-AsCpf1-puro vector with BamHI and EcoRI, and recover linearized pLVX-AsCpf1-puro vector by PCR Cleanup kit.

c. T4 DNA ligase was used to ligate a and b products. The ligated product was then transformed into E. coli Stbl3 and identified by sequencing. The recombinant vector contained in the correct strain was the pLVX-AsCpf1-puro-CNIH2 vector.

Examples 2 : Endotoxin-free plasmid DNA Preparation

The correct strain was sequenced and identified in Example 1 and placed in an LB liquid medium having an ampicillin concentration of 100 μg / ml, and cultured with shaking at 250 rpm and 37 ° C. for 12-16 h. Collect the bacterial solution by centrifugation at 10,000 rpm at 4 ° C, discard the supernatant, collect the bacterial cells, and then extract the plasmid according to the instructions of the Endo-Free Plasmid Mini Kit kit to obtain the endotoxin-free pLVX-AsCpf1-puro-CNIH2 plasmid.

Examples 3 : Packaging for Lentivirus

293T cells were cultured and transfected after 2 passages of growth and culture: pLVX-AsCpf1-puro-CNIH2 vector was taken and transfected with the packaging plasmid and transfection reagent provided by the integrase-deficient Lenti-X HTX lentivirus packaging system Stained in 293T cells. 48 hours before transfection, inoculate cells into a well plate or petri dish for lentivirus production. During transfection, the confluence of cells is about 70% -80% is the best infection state, and the viability is ≥95%. The staining time was the starting point. The supernatants were harvested after 48 h and 72 h, filtered through a 0.45 μm filter and stored at -80 ° C.

Examples 4 : RGC5 Lentiviral infection of cells and puromycin selection

When culturing RGC5 cells to a confluence of about 70% -80%, add a mixture of lentivirus and culture medium (containing 4 μg / mL After 24 hours of polybrene treatment, the lentivirus solution was changed to a complete medium containing 1 μg / mL puromycin, and the screening culture was started. The screening time was 7 days. Change the fluid every other day. Cells infected with lentivirus will form single-cell clones, and the cell selection is complete.

Examples 5 : T7 Endonuclease I Test to detect transduction effect

Untreated RGC5 cells (control group) and lentivirus-treated RGC5 cells (experimental group) were seeded into six-well plates. After the cells were full, genomic DNA was extracted, and then the high-fidelity PCR enzyme PrimeSTAR HS was used to expand The gene editing site is expected to be amplified, and the PCR product is recovered by electrophoresis.

After the PCR product was re-annealed, it was digested with T7 Endonuclease I at 37 ° C for 1 h, and then subjected to agarose gel electrophoresis. The results are shown in Figure 2. Compared with the control group, the experimental group showed two distinct cutting strips. The band indicates that the lentivirus packaged by the pLVX-AsCpf1-puro-CNIH2 vector can edit the human CNIH2 gene, indicating that the pLVX-AsCpf1-puro vector can be used in cell gene editing research.

Industrial applicability

The modified vector for human CNIH2 gene editing provided by the present invention has strong specificity, can edit human CNIH2 gene through the CRISPR / Cpf1 system very efficiently, and can be used in research and development of drugs related to abnormal expression of CNIH2 gene.

Claims (5)

1. A modified vector for human CNIH2 gene editing, characterized in that it comprises a core vector and an sgRNA that targets the human CNIH2 gene.
2. The modified vector for human CNIH2 gene editing according to claim 1, wherein the sequence of the sgRNA targeting the CNIH2 gene is 5'-AATTTCTACTCTTGTAGATCTGTCATCTGGCACGTAAGGCCG -3 ’.
3. The method for preparing a modified vector for human CNIH2 gene editing according to claim 2, characterized in that it comprises the following steps:

   (1) annealing and complementary pairing the sense and antisense strands of the sgRNA targeting the CNIH2 gene;

   (2) Digest the core vector with BamHI and EcoRI endonuclease, and recover the linearized core vector by agarose gel electrophoresis;

   (3) ligating the sgRNA obtained in step (1) to a linearized core vector with T4 DNA ligase;

   (4) The ligation product is transformed into competent E. coli Stbl3. After a large amount of culture, the recombinant vector is extracted and commissioned for sequencing. The correct sequencing result is the modified vector for human CNIH2 gene editing.
4. The method for preparing a modified vector for human CNIH2 gene editing according to claim 3, wherein in step (2) and step (3), the core vector is pLVX-ascpf1-puro.
5. The modified vector for human CNIH2 gene editing according to claim 1, wherein the vector is used for human CNIH2 gene knockout.