NoveSlice: A Novel Chromatin Context-Sensitive Gene-Editing Endonuclease

Mitchell Ryan Kopacz1, Franklin Kostas1, Jasmine K. Harris1,  Craig Mealmaker1, Christopher B. Rohde2, Matthew Angel2

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

Mol Ther, Vol 28, No 4S1, 2020

Genome-editing endonucleases are currently undergoing early clinical evaluation for the treatment of a wide range of diseases. However, in vivo use of gene-editing endonucleases is limited by the risk of potentially harmful off-target effects. It has been previously shown that gene-editing endonucleases are blocked by heterochromatin and show reduced efficiency in nucleosome-associated targets. We hypothesized that a gene-editing endonuclease with increased sensitivity to chromatin context could offer reduced off-target cutting without sacrificing on- target efficiency. Here we present NoveSlice, a novel heterodimeric gene-editing endonuclease comprising an engineered DNA-binding domain assembled from non-tandem repeat sequences connected by flexible linkers. We show that while both NoveSlice and TALENs are able to cleave a variety of targets with comparable efficiency in a cell- free amplicon-cutting assay, the efficiency with which NoveSlice cleaves genomic DNA depends more strongly on the chromatin context of the target than an equivalent TALEN pair. We constructed NoveSlice pairs targeting sequences within various human genes and the AAVS1 safe-harbor locus using both Golden Gate assembly and direct gene synthesis. We tested the effect of pair spacing and binding-site length on cutting efficiency both in a cell-free amplicon-cutting assay and, using a novel minimally immunogenic mRNA, in primary human epidermal keratinocytes, fibroblasts, induced pluripotent stem cells (iPSCs), and a cutaneous squamous cell carcinoma cell line. Both NoveSlice and TALENs efficiently cut a variety of targets in the AAVS1 safe-harbor locus in both the cell-free amplicon-cutting assay and in primary human cells. Also, co-transfection with a donor plasmid containing AAVS1 homology arms resulted in insertion of the donor into the AAVS1 locus. Similarly, both NoveSlice and TALENs efficiently cut a variety of targets in amplicons generated from genomic regions with reduced chromatin accessibility. However, while TALENs also efficiently cut these targets in primary human cells, NoveSlice showed no cutting of these targets in cells, suggesting that NoveSlice activity is more strongly dependent on the chromatin context of the target than an equivalent TALEN pair. The risk of potentially harmful off-target effects has restricted the clinical translation of gene-editing technologies. Here we present a novel gene-editing endonuclease that exhibits sensitivity to the chromatin context of the target. NoveSlice could thus serve as an important tool for the development of new precision medicines, including in vivo gene-editing therapies.