2. Academic Validation
  3. Small molecules enhance CRISPR/Cas9-mediated homology-directed genome editing in primary cells

Small molecules enhance CRISPR/Cas9-mediated homology-directed genome editing in primary cells

  • Sci Rep. 2017 Aug 21;7(1):8943. doi: 10.1038/s41598-017-09306-x.
Guoling Li 1 2,  Xianwei Zhang 1 2,  Cuili Zhong 1 2,  Jianxin Mo 1 2,  Rong Quan 1 2,  Jie Yang 1 2,  Dewu Liu 1 2,  Zicong Li 1 2,  Huaqiang Yang 3 4,  Zhenfang Wu 5 6
Affiliations

Affiliations

  • 1 National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, 510642, China.
  • 2 Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, 510642, China.
  • 3 National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, 510642, China. [email protected].
  • 4 Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, 510642, China. [email protected].
  • 5 National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, 510642, China. [email protected].
  • 6 Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, 510642, China. [email protected].
PMID: 28827551 DOI: 10.1038/s41598-017-09306-x
Abstract

CRISPR/Cas9 is an efficient customizable nuclease to generate double-strand breaks (DSBs) in the genome. This process results in knockout of the targeted gene or knock-in of a specific DNA fragment at the targeted locus in the genome of various species. However, efficiency of knock-in mediated by homology-directed repair (HDR) pathway is substantially lower compared with the efficiency of knockout mediated by the nonhomologous end-joining (NHEJ) pathway. Suppressing NHEJ pathway or enhancing HDR pathway has been proven to enhance the nuclease-mediated knock-in efficiency in cultured cells and model organisms. We here investigated the effect of small molecules, Scr7, L755507 and resveratrol, on promoting HDR efficiency in porcine fetal fibroblasts. Results from eGFP reporter assay showed that these small molecules could increase the HDR efficiency by 2-3-fold in porcine fetal fibroblasts. When transfecting with the homologous template DNA and CRISPR/Cas9 plasmid and treating with small molecules, the rate of knock-in porcine fetal fibroblast cell lines with large DNA fragment integration could reach more than 50% of the screened cell colonies, compared with 26.1% knock-in cell lines in the DMSO-treated group. The application of small molecules offers a beneficial approach to improve the frequency of precise genetic modifications in primary somatic cells.

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