Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) along with CRISPR Associated Protein 9 (CAS9) technology is now widely used in genome editing. CRISPR/CAS9 mediated gene editing uses guide RNA (sgRNA) to make a double stranded cut at the targeted gene sequence. Because the gene of interest is expected to be completely taken out of the genome, the effects of the knockout are able to be observed in all subdivided cells.<br/><br/>Morpholinos are used to knockdown targeted proteins of interest by blocking the translation the gene. Because this is not a change to the actual genome, however, this knockdown will only persist for a short amount of time due to cell division.<br/><br/>We are targeting DNA (cytosine-5)-methyltransferase 3A (DNMT3a) – an enzyme that can epigenetically regulate gene expression by adding methyl groups to DNA – during early chick eye formation. We are also targeting DNMT3a on five specific places with CRISPR/CAS9. The sgRNA vectors for CRISPR/CAS9 and DNMT3a morpholinos will be injected into the embryonic chick followed by electroporation at developmental stage 10. The morphogenic defects are apparent in the developing eye four days post-injection. The down regulation of DNMT3a can be assayed in the developing eye of the embryonic chick through western-blot and immunohistochemical staining three days post-injection in the case of CRISPR/CAS9 sgRNA and the morpholino treated groups. Using DNMT3a morpholinos, we have observed severe defects including microphthalmia (small eye) and ventral defects that result in failure to close the choroid fissure.

Knockdown and knockout of DNMT3a affects chick eye morphogenesis

251st National Meeting (2016)

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