Defects in the rhodopsin gene cause the most common form of the most common hereditary blinding disease, retinitis pigmentosa (RP) (1
). RP is a progressive neurodegenerative disorder that begins with the death of rod photoreceptors, where rhodopsin is expressed, but ultimately destroys rod and cone cells, leading to loss of both dim-light and color vision. All but a handful of the more than 100 rhodopsin alleles that cause retinal degeneration display dominant inheritance (1
). It is clear that dominance arises from the harmful effects of mutant rhodopsin, rather than from a lack of adequate protein levels, since human patients heterozygous for rare, null alleles of the rhodopsin gene and mice with one knockout allele do not suffer from serious degeneration (3–5
). Thus, it is the presence of rhodopsin encoded by the dominant allele that leads to degeneration, although it remains an open question whether its deleterious consequences are due to toxic gain-of-function or interfering dominant-negative effects (6
). In either case, correcting the defective allele, or knocking it out, would be expected to preserve retinal function by diminishing the loss of rod cells, which in turn would protect cones and extend the useful lifetime of central vision.
A promising general strategy for editing the genome in living cells is to introduce a double-strand break (DSB) into the target gene and exploit the normal cellular DSB repair processes to bring about the desired change (7–11
). Repair of the break by homologous recombination (HR), in the presence of a non-mutant segment of DNA to act as the template for repair, can correct the mutation (11
). Alternatively, repair by non-homologous end joining (NHEJ), which is usually mutagenic, can knockout gene expression (12
). Although HR and NHEJ have been well-defined in cycling mammalian cells (13
), their characterization in mature neurons, which no longer divide, is in its infancy (14
). At present, it is uncertain whether terminally differentiated rod photoreceptor neurons possess the requisite capabilities for DSB-dependent genome editing.
It is clear that the developing nervous system is exquisitely sensitive to DSBs, and that DSB repair by HR and NHEJ is critical to prevent cell death by apoptosis (15
). Defects in either repair pathway, or the signaling pathways that control them, can lead to neurological defects and disease in humans and mouse models (15–17
). For example, mutations in the ATM gene in humans lead to defects in the signaling response to DSBs, which causes ataxia telangiectasia, a progressive neurodegenerative disease that compromises the function of the cerebellum. Although HR and NHEJ play important roles in the proliferation and early stages of differentiation of the nervous system, their contribution to genome maintenance in the mature nervous system is poorly defined (14
). Because terminally differentiated, non-dividing nerve cells in the brain are mixed with a comparable number of glial cells that retain mitotic capability, analysis of DSB repair in neurons is inherently challenging. However, extracts from unfractionated and neuron-enriched rat brain samples display NHEJ activity, which declines with age (18
). These studies suggest that the NHEJ pathway of DSB repair is probably functional in mature neurons.
Studies in cycling cells, the source of virtually all our detailed information on DSB repair in mammalian cells, indicate that HR functions primarily in the S and G2 phases of the cell cycle, when a sister chromatid—the preferred template for HR repair—is available (20–22
). Since neurons are trapped in G1/G0, it is unclear a priori
whether HR is a viable strategy for modifying the rhodopsin gene in rod photoreceptors. The block to HR in G1 in cycling cells appears to be enforced at the step of 5′ to 3′ resection of the ends, which is required to expose the single-stranded DNA needed for the strand-invasion step in HR (23–25
). In contrast, end resection is not needed for NHEJ, which operates throughout the cell cycle (13
These considerations led us to expect that DSBs in rod photoreceptor cells would likely be repaired predominantly by mutagenic NHEJ and rarely, if at all, by HR. Thus, we designed a mouse model that would allow us to detect common NHEJ events by PCR-based methods and rare HR events by an exquisitely sensitive fluorescent assay that is capable of providing a reliable readout for one or a few events per retina (26
). To detect HR repair of DSBs, we replaced one mouse rhodopsin gene with a modified form of the human rhodopsin gene fused to enhanced GFP (26
). This modified rhodopsin-GFP gene carries two copies of exon 2, which flank the recognition sequence for the rare-cutting nuclease, ISceI. The duplicate copy of exon 2 shifts the reading frame so that rhodopsin-GFP is not expressed. Break-induced recombination between the duplicated segments will reconstitute a functional rhodopsin-GFP gene, giving rise to a bright fluorescent green outer segment on each corrected rod cell. This recombination substrate is expected to generate green rods by the single-strand annealing (SSA) pathway of HR. SSA, like the Rad51-mediated strand-invasion pathway of HR, begins by resection of ends at a DSB, but completes the repair event by pairing the exposed, single strands at complementary regions, thereby bypassing strand invasion. Thus, by assaying for the SSA pathway of HR, this duplication substrate tests whether end resection occurs during DSB repair in rod cells, which is a prerequisite for genome editing by HR.
Using recombinant adeno-associated virus (rAAV) to introduce the ISceI gene, we addressed three questions about DSBs in terminally differentiated rod photoreceptor neurons. Can DSBs be efficiently introduced into the rhodopsin gene? Can NHEJ and SSA repair DSBs in rod cells? And if so, what are their relative frequencies? Here, we show that most, if not all, rAAV-transduced rod cells were cleaved at the target site, with 85% of the DSBs repaired by NHEJ and 15% by SSA. The high frequency of DSB repair by mutagenic NHEJ lays the foundation for plausible therapeutic strategies designed to treat RP by knocking out dominant rhodopsin alleles in rod cells in patients. The surprisingly high frequency of DSB repair by SSA indicates that end resection is unlikely to block HR in rod cell neurons, raising the possibility that correction of mutant rhodopsin alleles by HR may also be achievable. Collectively, these results establish that the genomes of terminally differentiated rod photoreceptor neurons can be efficiently edited in living organisms.