DNA-binding domains containing novel repeat sequences enable temperature-tunable gene editing in primary human cells

Yemi Osayame1, Franklin Kostas2, Mitchell R. Kopacz2, Mackenzie Parmenter1, Christopher B. Rohde2, Matthew Angel2

1Novellus, Inc., Cambridge, MA, 2Factor Bioscience Inc., Cambridge, MA

Mol Ther, Vol 29, No 4S1, 2021

Sequence-specific gene-editing endonucleases, such as zinc finger nucleases (ZFNs), clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9), and transcription activator-like effector nucleases (TALENs) are being used in the development of many gene and cell therapies. However, these gene-editing endonucleases have seen limited in vivo application due to concerns about effects on genomic loci outside the intended cut site. TALENs consist of a DNA binding domain containing repeat-variable diresidues (RVDs) that confer site specificity fused to a nuclease catalytic domain. The RVDs contact individual bases in the target DNA sequence, and are connected by two alpha-helices linked by a short, disordered loop. We hypothesized that engineered gene-editing endonucleases comprising DNA-binding domains containing novel loops could exhibit different on-target cutting activity as well as altered specificity, due to the potential of this linkage to determine the degree of conformational disorder of the DNA-binding domain. We tested the ability of gene-editing endonucleases containing novel linkages to edit genes in primary cells, and discovered a striking temperature-dependence of gene-editing with certain linkages. We identified engineered endonucleases that efficiently edit only at sub-physiological temperatures, as well as endonucleases capable of high-efficiency editing in primary cells at 37 °C. We observed high-efficiency gene editing with the novel endonucleases in primary human dermal fibroblasts, primary epidermal keratinocytes, induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs). Additionally, we measured the specificity of the engineered endonucleases at various editing temperatures in cultured cells using LAM-PCR based sequencing. We show that engineering the RVD linkage of gene-editing endonucleases has the potential to improve on-target activity as well as sequence specificity in gene therapies.