Single-stranded DNA with internal base modifications mediates highly efficient knock-in in primary cells using...

Area of Science:

• Molecular Biology
• Gene Editing
• Genomics

Background:

• Single-stranded DNA (ssDNA) and Cas9 are used for genome knock-in but efficiency is often low.
• Chemical modifications to ssDNA can potentially improve gene editing outcomes.

Purpose of the Study:

• To investigate the efficacy of internally chemically modified ssDNA (esDNA) for enhancing genome knock-in efficiency.
• To compare esDNA performance against end-modified ssDNA in various cell types.

Main Methods:

• Development and application of enhanced ssDNA (esDNA) with 12-19% internal base chemical modifications.
• Testing esDNA for knock-in (KI) efficiency in airway basal stem cells (ABCs), CD34+ hematopoietic cells, T-cells, and endothelial cells.
• Investigating the role of TREX1 nuclease in esDNA-mediated KI, including knockout experiments.

Main Results:

• esDNA improved KI efficiency by 2-3 fold compared to end-modified ssDNA in multiple primary cell types.
• Achieved over 50% allele KI in clinically relevant loci (CFTR, HBB, CCR5) in ABCs.
• Demonstrated up to 70% allele KI in the HBB locus in CD34+ cells with a DNA-PKcs inhibitor.
• esDNA did not improve KI in induced pluripotent stem cells (iPSCs), potentially due to TREX1 absence.
• TREX1 knockout enhanced KI with unmodified ssDNA in other cell types.

Conclusions:

• Internally modified ssDNA (esDNA) represents a significant advancement for enhancing genome knock-in in primary cells.
• This method offers therapeutically relevant correction levels, comparable to AAV-based methods.
• The findings suggest TREX1 plays a crucial role in ssDNA-mediated gene editing, opening avenues for further optimization.
• esDNA is suitable for modifying small genomic regions (20-30 bp) in primary cells for therapeutic and research applications.