High-efficiency transgene integration by homology-directed repair in human primary cells using DNA-PKcs inhibition.
Sridhar SelvarajWilliam N FeistSebastien VielSriram VaidyanathanAmanda M DudekMarc GastouSarah J RockwoodFreja K EkmanAluya R OseghaleLiwen XuMara Pavel-DinuSofia E LunaM Kyle CromerRuhi SayanaNatalia Gomez-OspinaMatthew H PorteusPublished in: Nature biotechnology (2023)
Therapeutic applications of nuclease-based genome editing would benefit from improved methods for transgene integration via homology-directed repair (HDR). To improve HDR efficiency, we screened six small-molecule inhibitors of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key protein in the alternative repair pathway of non-homologous end joining (NHEJ), which generates genomic insertions/deletions (INDELs). From this screen, we identified AZD7648 as the most potent compound. The use of AZD7648 significantly increased HDR (up to 50-fold) and concomitantly decreased INDELs across different genomic loci in various therapeutically relevant primary human cell types. In all cases, the ratio of HDR to INDELs markedly increased, and, in certain situations, INDEL-free high-frequency (>50%) targeted integration was achieved. This approach has the potential to improve the therapeutic efficacy of cell-based therapies and broaden the use of targeted integration as a research tool.
Keyphrases
- high frequency
- genome editing
- circulating tumor
- small molecule
- endothelial cells
- crispr cas
- high efficiency
- cell free
- single molecule
- protein kinase
- single cell
- transcranial magnetic stimulation
- cell therapy
- dna repair
- induced pluripotent stem cells
- cancer therapy
- pluripotent stem cells
- copy number
- induced apoptosis
- nucleic acid
- high throughput
- dna damage
- genome wide
- cell cycle arrest
- drug delivery
- gene expression
- risk assessment
- cell death