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Method for improving gene knockout efficiency

A technology of gene knockout and high efficiency, which is applied in other methods of inserting foreign genetic materials, genetic engineering, plant gene improvement, etc., can solve the problem of low gene knockout efficiency and achieve the effect of improving gene knockout efficiency

Pending Publication Date: 2022-02-11
ZHUHAI HENGQIN IMSTEM BIOTECH LTD
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  • Summary
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Problems solved by technology

[0003] The purpose of the present invention is to solve the problem of low gene knockout efficiency in existing CRISPR / Cas9, and to provide a method for improving gene knockout efficiency

Method used

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  • Method for improving gene knockout efficiency
  • Method for improving gene knockout efficiency
  • Method for improving gene knockout efficiency

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Experimental program
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specific Embodiment approach 1

[0014] Specific embodiment one: In this embodiment, a method for improving gene knockout efficiency is carried out according to the following steps:

[0015] 1. Design 5-15 candidate sgRNAs for the target gene sequence fragments, and then synthesize them separately to obtain 5-15 groups of sgRNAs. Co-transfer each group of sgRNAs and Cas9 protein into cells, and extract each group 24h-48h after transfection The cellular genome of sgRNA is sequenced and analyzed to detect the gene editing efficiency of each candidate sgRNA;

[0016] 2. Screen sgRNAs with sgRNA concentration greater than 1ug / uL and editing efficiency greater than 30%, and select 3-6 sgRNAs in order of editing efficiency from high to low;

[0017] 3. Incubate 3-6 sgRNAs selected in step 2 with Cas9 protein in vitro to form a Cas9 / sgRNA protein complex;

[0018] 4. Mix the multiple Cas9 / sgRNA protein complexes assembled in step 3 and transfect them into the target cells;

[0019] 5. After the target cells are cu...

specific Embodiment approach 2

[0021] Embodiment 2: This embodiment differs from Embodiment 1 in that: the sequence fragment of the target gene is the first 50 bp of the target gene locus-the last 50 bp of the target gene locus. Others are the same as the first embodiment.

specific Embodiment approach 3

[0022] Embodiment 3: This embodiment differs from Embodiment 1 or Embodiment 2 in that: the sequence fragment of the target gene is the No. 58 enhancer sequence of the BCL11A gene. Others are the same as one of the specific embodiments 1 to 3.

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Abstract

The invention relates to a method for improving gene knockout efficiency, in particular to a method for improving gene knockout efficiency, and aims to solve the problem of low gene knockout efficiency of the existing CRISPR / Cas9. According to the invention, a gene editing cell transfection experiment is carried out by using a plurality of high-efficiency sgRNAs (about 3-6) in a sequence range of about 50bp upstream and downstream at the position of a target gene and combining with Cas9 protein, so that the gene knockout efficiency is remarkably improved, and the method is applied to the field of CRISPR-Cas9 mediated genome editing.

Description

technical field [0001] The present invention relates to a method for improving gene knockout efficiency. Background technique [0002] Compared with the traditional ZFN (zinc-finger nucleases) and TALEN (transcriptionactivator-like effector nucleases) gene editing technologies, CRISPR / Cas9 gene editing technology has been used in many gene therapy and life science researches due to its advantages of high efficiency and convenience. plays an important role. However, the existing CRISPR / Cas9 not only has off-target effects and sgRNA targeting position depends on the PAM site in the genome, but also has problems such as low gene knockout efficiency. If the knockout efficiency is low, the frequency of gene changes in cells after gene knockout will be low, and only very few cells have gene knockout. For the cell population, there is no obvious change in cell function, so the research on cell function and the change in cell function are greatly reduced. Contents of the inventi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/90C12N15/12
CPCC12N15/113C12N15/90C07K14/47C12N2310/20
Inventor 王小方孔明圣门增轩
Owner ZHUHAI HENGQIN IMSTEM BIOTECH LTD
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