Morpholino Mediated Gene Editing: A Gene Knockdown Technique Using Translational Blocking Morpholino Oligonucleotides in Single-Celled Cavefish Eggs

Morpholinos are short, single-stranded redesigned oligonucleotides with differences in their phosphate backbone and sugar structure as compared to natural nucleotides. These features enhance morpholinos’ stability at the physiological pH. Despite these variations, the morpholinos contain the standard nucleobases, which help in designing oligonucleotide complementary to the specific mRNA sequence from a desired target gene to be knocked down.

To perform morpholino-driven gene editing, begin by taking single-celled, cavefish eggs placed inside the wells of an agarose injection plate. Visualize the translucent and round eggs to locate the yolk sac - a fluid-filled structure present inside the egg.

Take a microinjection needle pre-filled with a solution containing designed morpholinos. Inject the solution directly into the egg yolk, ensuring morpholinos enter the cell cytoplasm. Inside the cells, the target gene forms the mRNA, which is transported to the cytoplasm.

Within the cytoplasm, the injected morpholino hybridizes with its complementary target mRNA transcript at the endogenous initiation site and extends beyond it. This process blocks the binding of the larger subunit of the ribosome with the nascent translation initiation complex consisting of small ribosomal subunit complexed with some specific proteins.

Subsequently, the progression of the translation initiation complex is sterically hindered, which stops the translation of mRNA into the protein. Finally, the target protein concentration reduces due to translational blocking by morpholinos.