Gene Editing Proteins with Nickase Functionality Enable Scarless Targeted Gene Insertion in Primary Human Cells

Elizabeth Belcher1, Taeyun Kim1, Kyle Garland1, Christopher B. Rohde2, Matthew Angel1,2

1Eterna Therapeutics Inc., Cambridge, MA, 2Factor Bioscience Inc., Cambridge, MA

Mol Ther, Vol 31, No 4S1, 2023

Many gene editing strategies involve the use of nucleases to generate targeted double-strand breaks (DSBs) in genomic DNA, which is often associated with cytotoxicity and off-target effects that can prevent clinical translation. Such undesirable outcomes have led to the development of gene-editing nickases, which instead create targeted single-strand breaks (SSBs) that favor high- fidelity repair through the homology-directed repair (HDR) pathway rather than the more error-prone non-homologous end joining (NHE]) pathway. Here, we explore the use of UltraSlice gene editing proteins containing cleavage domain variants with nickase functionality for targeted insertion of donor sequences into a defined genomic locus. Using UltraSlice gene editing proteins targeting exon 73 in COL7A1 (mutations in which cause dystrophic epidermolysis bullosa), we tested combinations of 3 mutations previously reported to confer nickase functionality to the catalytic domain of Fokl, a Type IIS restriction endonuclease (D450A, D450N, and D467A). Notably, we found that D450A and D450N resulted in significantly reduced NHEJ relative to the COL7A1_e73 UltraSlice pair with a wild-type FokI cleavage domain, with D450N exhibiting the least amount of NHE]. We confirmed these results with Sanger Sequencing, comparing the D450N-treated PCR amplicon to a wild-type COL7A1 PCR amplicon and observed no significant alteration of the genomic target site. We then compared the ability of nickases to insert a 300 bp dsDNA donor sequence via electroporation into primary human fibroblasts. Insertion band intensities showed high insertion efficiencies for D450A and D450N (58.7% and 42.9%, respectively), though lower than standard UltraSlice (72.3%). Our data demonstrate that gene-editing nickases enable scarless insertion of donor sequences into defined genomic loci, and thus may have the potential to improve the safety ofin vivo gene insertion by reducing off-target effects.

More Publications