Single-Stranded DNA with Internal Base Modifications Mediates Highly Efficient Gene Insertion in Primary Cells.
Karen L KankeRachael E RaynerEli AbelAparna VenugopalanMa SuuJacob T StackReza NouriGongbo GuoTatyana A VetterEstelle Cormet-BoyakaMark E HesterSriram VaidyanathanPublished in: bioRxiv : the preprint server for biology (2024)
Single-stranded DNA (ssDNA) templates along with Cas9 have been used for gene insertion but suffer from low efficiency. Here, we show that ssDNA with chemical modifications in 10-17% of internal bases (eDNA) is compatible with the homologous recombination machinery. Moreover, eDNA templates improve gene insertion by 2-3 fold compared to unmodified and end-modified ssDNA in airway basal stem cells (ABCs), hematopoietic stem and progenitor cells (HSPCs), T-cells and endothelial cells. Over 50% of alleles showed gene insertion in three clinically relevant loci ( CFTR, HBB , and CCR5 ) in ABCs using eDNA and up to 70% of alleles showed gene insertion in the HBB locus in HSPCs. This level of correction is therapeutically relevant and is comparable to adeno-associated virus-based templates. Knocking out TREX1 nuclease improved gene insertion using unmodified ssDNA but not eDNA suggesting that chemical modifications inhibit TREX1. This approach can be used for therapeutic applications and biological modeling.
Keyphrases
- genome wide
- copy number
- stem cells
- highly efficient
- genome wide identification
- endothelial cells
- dna damage
- crispr cas
- induced apoptosis
- cystic fibrosis
- dna methylation
- cell free
- cell proliferation
- signaling pathway
- transcription factor
- circulating tumor
- binding protein
- endoplasmic reticulum stress
- mesenchymal stem cells
- genome wide analysis
- oxidative stress
- cell death
- regulatory t cells