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Method for increasing non-homologous end joining efficiency of CRIPSR/Cas9 target knock-out genes

A gene and efficiency technology, applied in the field of improving the efficiency of non-homologous end joining of CRIPSR/Cas9 targeted knockout genes, can solve the problems of reducing the fidelity of the repair process and gene breakage

Inactive Publication Date: 2017-06-30
重庆高圣生物医药有限责任公司
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Problems solved by technology

These oligonucleotides may reduce the fidelity of this repair process, or switch the cell to a repair process that occurs more readily, allowing Cas9 to break genes more easily

Method used

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  • Method for increasing non-homologous end joining efficiency of CRIPSR/Cas9 target knock-out genes
  • Method for increasing non-homologous end joining efficiency of CRIPSR/Cas9 target knock-out genes
  • Method for increasing non-homologous end joining efficiency of CRIPSR/Cas9 target knock-out genes

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Embodiment Construction

[0013] The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. For the experimental methods that do not specify specific conditions in the examples, generally follow the conventional conditions, such as the conditions described in the Molecular Cloning Experiment Guide (Third Edition, J. Sambrook et al.), or the conditions suggested by the manufacturer.

[0014] According to the NCBI database, the CDS region of the gene PTEN was discovered, and the primer sequence was designed in combination with NCBI Primer-BLAST. At the same time, according to the patent (201611252179.5), the primer for the amplification of the target site region of the lin28a gene was synthesized. The sequence is as follows:

[0015]

[0016] Five small interfering RNA sequences targeting PTEN gene were designed and synthesized by online software, and the corresponding RNA sequences are shown in SEQ ID NO.1--SEQ ID NO.5.

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Abstract

The invention belongs to the technical field of molecular biology and biomedicine, and relates to a method for increasing the efficiency of CRIPSR / Cas9 target knock-out genes NHEJ. According to the method for increasing the efficiency of CRIPSR / Cas9 target knock-out gene NHEJ, small molecule interference RNA that efficiently interferes with expression of PTEN genes is screened out; by using CRISPR / Cas9 system to target knockout genes, the small molecule RNA is cotransfected into the target gene, thus the NHEJ efficiency of target genes is effectively improved and the NHEJ of target gene in cell multiple targets is significantly improved. The method has the advantages of low cost, simple operation and high efficiency, and has a good promoting effect on the efficiency and application of CRISPR / Cas9 technology.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and biomedicine, and in particular relates to a method for improving the efficiency of non-homologous end joining (NHEJ) of CRIPSR / Cas9 targeted knockout genes. Background technique [0002] The CRISPR / Cas system is developed from the adaptive immune system of bacteria and archaea against foreign viruses or plasmids, including three different types, of which the DNA endonuclease Cas9 of the Type Ⅱ CRISPR / Cas system has only one subclass. The base has the simplest structure, so it is the most widely used. In addition to the Cas9 protein, the system also includes two short CRISPR RNAs (crRNAs) and trans-activating crRNAs (tracrRNA). The mature crRNA-tracrRNA complex can guide the Cas9 protein to the target sequence through complementary base pairing, and specifically cut the DNA double strand near the PAM (protospace radjacent motif) to form a DSB (double strand break). DSB can be repai...

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Application Information

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IPC IPC(8): C12N15/87C12N15/85C12N15/113
CPCC12N15/1137C12N15/85C12N15/87C12N2310/14C12N2810/10
Inventor 周勇申友锋
Owner 重庆高圣生物医药有限责任公司
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