Method for improving efficiency of homologous repair in CRISPR/CAS9 gene editing
A technology for gene editing and repair efficiency, applied in the field of gene editing, can solve the problems of low success rate of gene editing individuals, low proportion of homozygous homozygotes for homologous repair, difficulty in positive cell selection, etc., and achieves simple operation, low cost and obvious effect Effect
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[0026] Example 1 Method for Improving Homologous Repair Efficiency in CRISPR / CAS9 Gene Editing
[0027] This example takes the repair of myostatin (MSTN) gene as an example, and provides a method for improving the efficiency of homologous repair in CRISPR / CAS9 gene editing.
[0028] 1. MTR calculation method
[0029] 10 cultured in vitro for in vitro transfection 6 cell as an example, the MSTN allele content is 0.34×10 - 5 pMol, if the MTR value is 5 × 10 5 , then the repair template DNA (homology arm construct) is 1.7 pMol. If the repair template DNA contains carrier redundant sequences, only calculate the molecular weight of the DNA including the homology arms, and then calculate the amount (pg or pM) of the repair template DNA that needs to be added.
[0030] 2. In vitro gene editing and homologous repair of fetal fibroblasts
[0031] 2.1 Culture of fetal fibroblasts in vitro
[0032] Fetuses of Laoshan dairy goat, Landrace pig, Jinnan cattle, etc. were collected for...
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