Purchase this article with an account.
MANEL LLADO SANTAEULARIA, Federica Esposito, Carolina Iodice, Elena Marrocco, Alberto Auricchio; Homology-Independent Targeted Integration in Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4228.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Retinitis pigmentosa (RP) is a heterogeneous group of inherited ocular diseases affecting 1/3.000-5.000 people worldwide. Thirty-40% of all cases of RP have an autosomal dominant inheritance. Mutations in the rhodopsin gene (RHO) are responsible for 20% of cases of dominant RP in the world. Genome editing strategies for dominantly inherited diseases are limited by the need to knock out the mutant allele without affecting the wildtype allele. Gene correction using homologous recombination is generally inefficient in differentiated neurons. Homology-Independent Targeted Integration (HITI) has recently been developed to overcome both challenge1. HITI allows precise integration of a donor DNA template in a site of interest by CRISPR-Cas9 cleavage, followed by non-homologous end joining-mediated integration of the DNA template. We have adapted this system with the aim of developing a gene editing approach that can be used in an allele-independent manner.
We have designed a gRNA specific for mouse Rho (mRHO) exon 1, and used dsRED as a donor DNA for proof of concept studies. We have used a scramble gRNA as a negative control. We transfected HEK293 cells with a plasmid expressing mRHO, a plasmid expressing Cas9-GFP and the gRNA, and a plasmid carrying the donor DNA.We used subretinal injection to deliver 2,5*109 genome copies of each of two AAV8 vectors, encoding Cas9 and the donor DNA respectively, in the retina of 4-week old C57BL/6 mice.We applied this system also in the pig retina by using a pig-specific gRNA and donor DNA-flanking region, injecting 2*1011 genome copies of each AAV8.
In HEK293 cells co-transfected with a template CMV-mRho plasmid, Cas9 cleaves both the target locus and the donor DNA, and integration occurs in 66% of transfected cells, with surprising precision. Thirty days post-injection we observed up to 9,1% efficiency of dsRED integration in mouse rod photoreceptors, only when the donor DNA is delivered together with mRho specific gRNA.Thirty days after subretinal injection, we observed dsRED+ photoreceptors in pig retinas treated with gRNA and not scramble.
HITI is a viable and effective option for genome editing in the retina of mice and large animal models. We are currently adapting this system to integrate a correct copy of the RHO coding sequence in one or both alleles of a mouse model of autosomal dominant RP to obtain simultaneously both RHO knock-out and replacement.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
This PDF is available to Subscribers Only