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CN-122012629-A - HDR enhancer for improving cell homologous recombination efficiency and application thereof

CN122012629ACN 122012629 ACN122012629 ACN 122012629ACN-122012629-A

Abstract

The invention relates to the technical field of genetic engineering, in particular to an HDR enhancer for improving the cell homologous recombination efficiency and application thereof, wherein the HDR enhancer comprises an inhibitor of a non-homologous end-connection repair pathway and an intracellular homologous recombination promoter, wherein the inhibitor of the non-homologous end-connection repair pathway is SCR-7 and M3814, the intracellular homologous recombination promoter is L755507 and Romidepsin, and the four are used for cooperatively targeting an NHEJ signal pathway, a cell cycle, opening of a chromatin structure and simultaneous regulation of the HDR signal pathway, so that the problem of insufficient synergistic effect of the existing reagent for improving the cell homologous recombination efficiency is solved.

Inventors

  • Yuan Dandong
  • ZHENG HONG
  • Liang Zechuan
  • ZHENG DANDAN
  • He Yingxing
  • YE HAIFENG

Assignees

  • 广州源井生物科技有限公司

Dates

Publication Date
20260512
Application Date
20260204

Claims (8)

  1. 1. An HDR enhancer for improving the efficiency of homologous recombination in cells, characterized by comprising an inhibitor of a non-homologous end joining repair pathway and an intracellular homologous recombination promoter; Inhibitors of the non-homologous end joining repair pathway are SCR-7 and M3814, and the intracellular homologous recombination promoters are L75507 and Romidepsin.
  2. 2. The HDR enhancer for improving the efficiency of cell homologous recombination according to claim 1, wherein SCR-7 has a working concentration of 1 to 20. Mu.M, romidepsin has a working concentration of 0.001 to 0.2. Mu.M, M3814 has a working concentration of 0.1 to 10. Mu.M, and L7555507 has a working concentration of 0.1 to 20. Mu.M.
  3. 3. The HDR enhancer for improving the efficiency of cell homologous recombination according to claim 1, wherein the HDR enhancer consists of SCR-7, romidepsin, M3814 and L755507; Wherein the working concentration of SCR-7 is 1-20uM, the working concentration of Romidepsin is 0.001-0.2uM, the working concentration of M3814 is 0.1-10 uM, and the working concentration of L7555507 is 0.1-20uM.
  4. 4. An HDR enhancer for improving the efficiency of cellular homologous recombination according to claim 3, wherein the HDR enhancer consists of SCR-7, romidepsin, M3814 and L755507; wherein, the working concentration of SCR-7 is 5uM, the working concentration of Romidepsin is 0.1 uM, the working concentration of M3814 is 5uM, and the working concentration of L7555507 is 20uM.
  5. 5. A method of increasing the efficiency of cellular homologous recombination using the HDR enhancer of any one of claims 2-4, comprising the steps of: S1, adding an HDR enhancer into a complete culture medium according to the volume of the complete culture medium required and the corresponding working concentration requirement to prepare a cell culture medium containing the HDR enhancer; S2, preparing a premix compound for gene editing, mixing the premix compound with cells for electrotransformation, and transferring the cells after electrotransformation to a cell culture medium containing an HDR enhancer for culture.
  6. 6. The method for improving the efficiency of homologous recombination according to claim 5, wherein in step S2, the steps before the culturing are as follows: (1) Culturing the cells to 70-80% confluence, pouring out the culture medium, and digesting and counting the cells by using pancreatin; (2) Taking 3×10 5 -4×10 5 cells into a centrifuge tube, centrifuging, and discarding supernatant to obtain centrifuged cells; (3) Taking another centrifuge tube, adding the required sgRNA, donor template and cas9 protein, adding an electrotransfer buffer solution to complement to 10ul, uniformly mixing to obtain the premix compound, and standing for later use; (4) And fully mixing the premixed compound with the centrifuged cells, and setting electrotransformation parameters for electrotransformation.
  7. 7. The method of claim 5, wherein in step S1, the prepared HDR enhancer-containing cell culture medium is preheated in an incubator at 37 ℃.
  8. 8. The method of claim 6, wherein in the step (2), the centrifugation is performed at 300g for 3min.

Description

HDR enhancer for improving cell homologous recombination efficiency and application thereof Technical Field The invention relates to the technical field of genetic engineering, in particular to an HDR enhancer for improving the homologous recombination efficiency of cells and application thereof. Background The DNA repair mechanism mainly comprises non-homologous end joining (non-homologous DNA end joining, NHEJ) and homologous recombination (homologous DIRECTED REPAIR, HDR), wherein the non-homologous end joining is a rapid repair mode which directly bonds broken genomes together without depending on DNA Homology, the repair mode is relatively efficient and can occur at any period of cell growth, the homologous recombination is an accurate broken DNA repair method, the homologous DNA is required to be used as a template for repair, and the repair generally only occurs in the S-G2/M phase of cells, so that the cell editing efficiency is low. Cell gene point mutation technology or gene fixed-point knock-in technology developed based on CRISPR/Cas9 system provides a specific homologous recombination sequence after genome double-strand break is caused by fixed-point cutting of DNA, so that the cell repairs the DNA double-strand break in a homologous recombination mode, and simultaneously, a required fragment is inserted into the genome, however, the probability and efficiency of homologous recombination are low, and the construction success rate of the point mutation or gene knock-in cell is low. For this reason, the current methods for improving the efficiency of homologous recombination mainly include inhibition of NHEJ occurrence, cell cycle arrest or promotion of chromatin structure opening, etc. Many agents for cell cycle regulation and chromatin modification have insufficient synergistic effects, and are inherently more cytotoxic or mutagenic, which can lead to decreased cell viability and even cause non-specific gene damage, affecting subsequent gene editing efficiency and mutant cell construction. Disclosure of Invention Aiming at the defects, the invention aims to provide an HDR enhancer for improving the cell homologous recombination efficiency and application thereof, and solves the problem that the existing agent for improving the cell homologous recombination efficiency has insufficient synergistic effect. In order to achieve the purpose, the invention adopts the following technical scheme: An HDR enhancer for improving the efficiency of cellular homologous recombination, comprising an inhibitor of a non-homologous end joining repair pathway and an intracellular homologous recombination promoter; Inhibitors of the non-homologous end joining repair pathway are SCR-7 and M3814, and the intracellular homologous recombination promoters are L75507 and Romidepsin. Preferably, SCR-7 has an operating concentration of 1-20uM, romidepsin has an operating concentration of 0.001-0.2uM, M3814 has an operating concentration of 0.1-10 uM, L7555507 has an operating concentration of 0.1-20uM. Preferably, the HDR enhancer consists of SCR-7, romidepsin, M3814, and L755507; Wherein the working concentration of SCR-7 is 1-20uM, the working concentration of Romidepsin is 0.001-0.2uM, the working concentration of M3814 is 0.1-10 uM, and the working concentration of L7555507 is 0.1-20uM. Further, the HDR enhancer consists of SCR-7, romidepsin, M3814, and L755507; wherein, the working concentration of SCR-7 is 5uM, the working concentration of Romidepsin is 0.1 uM, the working concentration of M3814 is 5uM, and the working concentration of L7555507 is 20uM. A method of increasing the efficiency of cellular homologous recombination using an HDR enhancer of any of the above, comprising the steps of: S1, adding an HDR enhancer into a complete culture medium according to the volume of the complete culture medium required and the corresponding working concentration requirement to prepare a cell culture medium containing the HDR enhancer; S2, preparing a premix compound for gene editing, mixing the premix compound with cells for electrotransformation, and transferring the cells after electrotransformation to a cell culture medium containing an HDR enhancer for culture. Preferably, in step S2, the steps before culturing are: (1) Culturing the cells to 70-80% confluence, pouring out the culture medium, and digesting and counting the cells by using pancreatin; (2) Taking 3×10 5-4×105 cells into a centrifuge tube, centrifuging, and discarding supernatant to obtain centrifuged cells; (3) Taking another centrifuge tube, adding the required sgRNA, donor template and cas9 protein, adding an electrotransfer buffer solution to complement to 10ul, uniformly mixing to obtain the premix compound, and standing for later use; (4) And fully mixing the premixed compound with the centrifuged cells, and setting electrotransformation parameters for electrotransformation. Preferably, in step S1, the cell culture medium containing the HDR enhancer p