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Blake J Rasor, Brad D Wagner, Katia Del Rio-Tsonis, Michael L Robinson; High efficiency CRISPR/Cas9-mediated insertion of a fluorescent reporter into the mouse Mitf locus in ESCs. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1772.
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© ARVO (1962-2015); The Authors (2016-present)
Ground breaking work by the Sasai laboratory demonstrated that mouse embryonic stem cells (ESCs) can form self-organizing optic cups in three-dimensional culture (Eiraku et al., 2011 Nature 472:51-56). The EB5 ESC line created in the course of these experiments expresses GFP in the early eye field as the result of a conventionally generated knock-in of GFP at the Rax locus. Although it is very useful for visualization of early eye field differentiation, this particular ESC line, from inbred strain 129/Ola, exhibits reduced pigmentation of retinal pigment epithelial (RPE) cells due to homozygosity for a tyrosinase mutation. Here we describe a CRISPR/Cas9 strategy to insert an mCherry reporter into the Mitf locus to mark early RPE differentiation without compromising the function of MITF.
CRISPR/Cas9-mediated gene editing introduced a double strand DNA break following the stop codon of Mitf, which was repaired via homology-directed repair (HDR). The HDR template consisted of mCherry and a FRT-flanked Neo cassette for positive selection flanked by 5’ and 3’ homology arms of 1.5 Kb each. A P2A sequence was included at the 5’ end of mCherry to facilitate cleavage of the fluorophore from MITF during translation to avoid inhibiting the function of either protein. The gene for herpes simplex virus thymidine kinase (HSV-TK) was placed after the 3’ UTR of Mitf in the HDR template for negative selection. EB5 ESCs were electroporated with the HDR template and the CRISPR plasmid containing Cas9 and the guide RNA to direct the nuclease to Mitf. The antibiotics G418 and ganciclovir selected for Neo and against HSV-TK, respectively.
57% of the 96 tested clones surviving selection displayed the properly edited Mitf locus, as verified by PCR screening and DNA sequencing. This represents a substantial increase in ESC targeting efficiency relative to traditional homologous recombination strategies. Selected clones are currently being tested via in vitro eye cup formation and in chimeric mice to ensure the anticipated onset of GFP expression in the eye field followed by mCherry expression in the early RPE.
CRISPR/Cas9-mediated HDR effectively mediated the insertion of a large DNA fragment (~2.6 Kb) into a specific site within the mouse MitF locus. These ESCs should facilitate simple visualization of both early eye field and early RPE differentiation in vitro and in vivo.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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