Differences in the Response to DNA Double-Strand Breaks between Rod Photoreceptors of Rodents, Pigs, and Humans.

Florian Frohns,Carla Rohrer-Bley,Markus Löbrich,Antonia Frohns,Johanna Kramer,Irina Solovei,Katharina Meurer,David Hicks,Paul G Layer

doi:10.3390/cells9040947

Abstract

Genome editing (GE) represents a powerful approach to fight inherited blinding diseases in which the underlying mutations cause the degeneration of the light sensing photoreceptor cells of the retina. Successful GE requires the efficient repair of DNA double-stranded breaks (DSBs) generated during the treatment. Rod photoreceptors of adult mice have a highly specialized chromatin organization, do not efficiently express a variety of DSB response genes and repair DSBs very inefficiently. The DSB repair efficiency in rods of other species including humans is unknown. Here, we used ionizing radiation to analyze the DSB response in rods of various nocturnal and diurnal species, including genetically modified mice, pigs, and humans. We show that the inefficient repair of DSBs in adult mouse rods does not result from their specialized chromatin organization. Instead, the DSB repair efficiency in rods correlates with the level of Kruppel-associated protein-1 (KAP1) expression and its ataxia-telangiectasia mutated (ATM)-dependent phosphorylation. Strikingly, we detected robust KAP1 expression and phosphorylation only in human rods but not in rods of other diurnal species including pigs. Hence, our study provides important information about the uniqueness of the DSB response in human rods which needs to be considered when choosing model systems for the development of GE strategies.

Highlights

Retinal dystrophies which are caused by inherited genome mutations can result in the degeneration of rod and cone photoreceptors (PRs) as the light-sensing cells of the retina
We previously described a substantial double-stranded breaks (DSBs) repair defect in rod PRs of adult mice which was not observed in undifferentiated rods at an early postnatal stage (P4)
While the time during development at which the nuclear inversion in rods occurs has already been investigated in previous studies [20], the onset time of the emerging defects in Kruppel-associated protein-1 (KAP1) signaling and DSB repair remained unknown

Summary

Introduction

Retinal dystrophies which are caused by inherited genome mutations can result in the degeneration of rod and cone photoreceptors (PRs) as the light-sensing cells of the retina. In order to make use of the higher accuracy of HR over NHEJ, there are efforts to establish high fidelity GE by HR even in postmitotic cells. This can be achieved by the combined delivery of a donor template together with the CRISPR-associated endonuclease Cas9 [5]. Using this approach, Bakondi et al could successfully modify mutated alleles in postmitotic rods of newborn pups of a rat model for rod degeneration-based blinding diseases [6]. Various research groups are working on ways to further improve the HR efficiency in postmitotic cells, e.g., by using molecules that are inhibiting NHEJ [7]

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