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Construction and application of adenovirus-associated viral vector of CRISPR / Cas9 endonuclease system

A viral vector, virus technology, applied in the field of genes, can solve the problems of less expression and insertion mutation

Active Publication Date: 2016-10-19
CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, lentivirus will insert into the genome to cause insertion mutation; while adenovirus is type 5 adenovirus, which is hepatotropic and has very little expression in other tissues

Method used

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  • Construction and application of adenovirus-associated viral vector of CRISPR / Cas9 endonuclease system
  • Construction and application of adenovirus-associated viral vector of CRISPR / Cas9 endonuclease system
  • Construction and application of adenovirus-associated viral vector of CRISPR / Cas9 endonuclease system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0109] The construction of the AAV expression plasmid of embodiment 1, Cas9

[0110] The key element ITR of AAV expression vector (pAAV-MCS) is retained, and the middle sequence of ITR is replaced by CMV promoter and minipolyA site. Obtain 3×Flag-NLS-Cas9-NLS sequence from pX330 vector and insert it between CMV and minipolyA sites to obtain AAV-Cas9, in which the ITR intermediate sequence is 5.0kb in total, and the schematic diagram of its main components is as follows figure 1 A, designed so that AAV package size constraints can be met.

[0111] Construct the corresponding sgRNA expression vector Insert the chimeric RNA expression element in pX330 into the AAV expression vector (pAAV-MCS) and replace the ITR intermediate sequence to obtain AAV9-sgRNA. The schematic diagram of the main elements is as follows figure 1 b.

Embodiment 2

[0112] Example 2, Cas9 was successfully packaged into AAV9 adeno-associated virus and successfully expressed in mice

[0113] The constructed AAV expression vector of Cas9 (AAV-Cas9) was packaged into AAV9 adeno-associated virus, and intracardiacly injected into neonatal mice (within one week). One month later, different tissues of the mice were harvested, and the expression of Cas9 protein was detected by western blot after homogenization.

[0114] The result is as figure 2 As shown, Cas9 expression was highest in the heart, followed by brain and muscle. Other tissues expressed very low or undetectable.

Embodiment 3

[0115] Example 3. The Cas9 protein expressed through AAV9 can edit the DNA of cardiomyocytes under the combined action of sgRNA and tracrRNA

[0116] AAV9 adeno-associated virus expressing Cas9 and sgRNA chimeric RNA was injected into the left ventricle of neonatal mice, and genomic DNA of mouse myocardium was extracted one month later. The sequence of the DNA segment is detected by amplifying the DNA segment targeted by the sgRNA for sequencing. At the same time, Surveyor nucleic acid mismatch enzyme was used to detect the ratio of mutations in DNA.

[0117] The result is as image 3 As shown in A-B, mice injected with AAV9 virus expressing Cas9 and sgRNA had mutations in the target DNA of the myocardial genome, and these were insertion or deletion mutations. Mutation efficiency was then tested using Surveyor mismatch endonuclease. Genomic target DNA fragments are amplified using PCR and subjected to denaturation and reannealing slowly and uniformly, at which point wild-ty...

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Abstract

The invention relates to construction and application of an adenovirus-associated viral vector of a CRISPR / Cas9 endonuclease system. Through reduction and optimization of a Cas9 expression element, an AAV expression vector of a Cas9 protein with tissue cell broad-spectrum expression is prepared, and the entire expression element and Cas9 encoding sequence are packaged into the AAV virus for the first time. Based on the characteristics of AAV virus packaging, the Cas9 vector can be packaged into AAV virus of different serotypes by only replacing a packing capsid plasmid. The virus obtained by the invention can effectively realize the targeting expression of tissue and DNA editing.

Description

technical field [0001] The invention belongs to the field of gene technology, and more specifically, the invention relates to the construction of an adeno-associated virus vector of a CRISPR / Cas9 endonuclease system and its application. Background technique [0002] The type II prokaryotic CRISPR / Cas system is an acquired immune system of bacteria capable of degrading phage DNA or foreign plasmids. The modified artificial endonuclease CRISPR / Cas9 consists of Cas9 protein, crRNA and tracrRNA containing guide sequences. Through the 20-base guide sequence and the complementary pairing combination of the target DNA, Cas9 cuts the target DNA to generate a double bond break. DNA double-strand breaks are repaired by high-fidelity homologous recombination or non-homologous end-joining pathways that easily introduce insertion / deletion mutations. When repairing by homologous recombination, in the presence of a homologous template, directional repair will be carried out according to ...

Claims

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Application Information

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IPC IPC(8): C12N15/864C12N15/55C12N7/01
Inventor 宋保亮谢畅
Owner CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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