Purchase this article with an account.
Miruna Ghinia, Kevin Gonzalez, Mark Emerson; CRISPR-mediated Otx2 and OC1 KO in the chicken retina. Invest. Ophthalmol. Vis. Sci. 2017;58(8):125.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The precise manner by which retinal cells are born in a successive, yet overlapping sequence is still an open question. One powerful approach to elucidate the mechanisms of diversification in the developing retina is through investigation of the gene regulatory networks, specifically in the context of mutations in key developmental genes. Genes like Otx2 and OC1 are proven to be involved in promoting the cone fate while repressing the rod fate in both chicken and mouse. Here we describe a method to permanently ablate these genes and simultaneously insert reporter genes into the locus in the chick genome using a CRISPR strategy.
We use a two-step, recombinase-based method to knock in reporter genes at the Otx2 and OC1 loci. Chicken embryos at Embryonic day 1.5 are electroporated in-vivo with constructs carrying the Cas9 gene, guide RNA and a single stranded targeting DNA oligo that provides a first recombination site to be knocked in. Three days later, the retinas are removed and an ex-vivo electroporation is performed with a plasmid harboring the second recombination site followed by either GFP or mCherry, along with a plasmid carrying the recombinase protein. Upon recombination of the two sites, the fluorescent proteins will be expressed under the Otx2 or OC1 promoters.
We confirmed the successful knock-in of the first recombination site by genotyping PCR, which is illustrated by increasing intensity of the knock-in band as the concentration of the ssDNA targeting oligo delivered to the retina was raised. Secondly, we used immunohistochemistry to confirm the mutant phenotypes of the different genes targeted by CRISPR in the retina, which were disrupted upon RNA guide-directed Cas9 double stranded break. Finally, we were able to efficiently knock in fluorescent proteins at the desired location in the genome, allowing for a better characterization of the mutant phenotypes, with regards to the morphology of the cells affected and their overall impacts to the developing retina.
Using electroporation as a fast and reliable delivery method, we implemented the bacterial Cas9 to target and modify genes that are essential during retinal development. These experiments have not only demonstrated that the CRISPR-Cas9 system is suitable for genomic modifications in the chick retina, but they have also enabled us to study the genetic mechanisms that regulate retinal development in this organism.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
This PDF is available to Subscribers Only