ESGCT

Presentation: A lightbulb moment for gene editing

Abstract

CRISPR-Cas9 offers precise genome targeting and significantly reduced costs and biosafety risks compared to viral vectors for genetic engineering. CRISPR relies on donor DNA as a template for homology-directed repair (HDR) to introduce sequences of choice into the genome for cell reprogramming. Generally, single-stranded DNA (ssDNA) is more efficient, less toxic, and more specific than double-stranded DNA (dsDNA) when used as HDR template for CRISPR gene editing. However, production of long single-stranded oligodeoxynucleotides (ssODN) is difficult and costly. That is why non-viral CRISPR gene editing still largely relies on plasmid DNA as the donor for gene-length insertions, despite the high immunogenicity, low knock-in efficiencies and off-target effects associated with it. One of Touchlight’s novel platforms is megabulb DNA (mbDNA) – a closed synthetic single-stranded CRISPR HDR donor. Its elegant design combined with Touchlight’s manufacturing technology promises increased insert size and improved specificity to existing genome editing technologies. We have shown that mbDNA mediates gene-length knock-ins more efficiently than other DNA donor templates, such as ssODN and plasmid. Importantly, it exhibits extremely low primary T-cell toxicity even at high concentrations, allowing the introduction of increased template amounts to significantly improve the live knock-in cell counts produced with dsDNA donors. Moreover, mbDNA shows more consistent immunogenicity profiles and editing efficiencies across blood donors, which makes it a safer and more reliable CRISPR template of choice, particularly in autologous gene therapy. Therefore, mbDNA is addressing many of the current limitations in CRISPR gene editing and, in this way, promising to advance non-viral gene therapy.