Scientists edit the genome through utilizing naturally occurring processes of gene regulation, and one key component of that is RNA. In regulating the genome, cells turn on and off particular regions of DNA, reduce the rate of translation of DNA into proteins, and more. Each process requires the use of RNA molecules.
For the gene editing toolkit, there are RNA molecules that scientists should be aware of; thesearesgRNA, shRNA, siRNA, and miRNA.
Sample preparation is necessary for the majority of life science research experiments; enabling compatibility and accuracy while preventing interference from contaminants for successful analysis.
Initial steps include DNA or RNA Extraction (which can include the use of Columns & Beads), Protein Purification and Immunoprecipitation for isolation of the target molecule.
Sample cleanup and enrichment kits go one step further to increase concentration and remove any remaining impurities which may prevent proper analysis.
Sample preservation allows the prepared target molecule to be stabilized and stored until required; preventing degradation in the process.
Short Hairpin RNA, or shRNA, are synthetic RNA molecules with a hairpin loop used to silence gene expression. A hairpin loop structure is formed when the base pairs of a strand of RNA binds to itself, forming a U-like shape. shRNA is delivered to target cells via plasmids or vectors, which then incorporate into the DNA and are reproduced in the target cell. They then follow the same RNAi pathway via RISC as previously described. However, given the fact that shRNA is integrated into the target cell’s DNA, shRNA are constantly produced, thus providing long-lasting gene silencing.
Small interfering RNA (siRNA), is a double strand of RNA ranging in 20 – 25 of base pairs long whose primary function is to repress transcription, thus silencing genes. Once siRNA enters a cell, it is incorporated into other proteins to form RISC, the RNA-induced Silencing Complex. Then, siRNA is unraveled into a single strand of RNA, which goes on to find a complementary strand of mRNA. Once binding between these two RNAs is initiated, siRNA triggers a cleavage event, thus deactivating the mRNA and reducing the rate of translation. One drawback to siRNA is its fragility and is degraded in target cell’s cytoplasm. Thus, it is necessary to deliver a continuous high dosage, if siRNA’s are to be used for gene therapy.
microRNA (miRNA) are small noncoding RNA molecules, usually 22 nucleotides long, and are involved in RNA silencing and post-transcriptional regulation. miRNAs are similar to siRNA and shRNA in that they participate in silencing pathways, but differ in how they are manufactured. miRNAs are transcribed by the host cell’s DNA and form short hairpin loops within the nucleus. Once exported into the cytoplasm, the Dicer enzyme then removes the hairpin loop structure allowing for the miRNA double strand to unwind. This new single strand of miRNA then pairs with its complementary mRNA strand where it either initiates cleavage or blocks translation.