guide RNA Manufacturing

Gene editing is a process that takes advantage of the natural repair system of DNA strands in order for specific changes to be made in the DNA. Possibly the most well-known platform of gene editing is the Cas9 system that uses sgRNA (single guide RNA). This system was pioneered by Emmanuelle Charpentier and Jennifer A. Doudna, who won the Nobel Prize in Chemistry in 2020 for their work in recreating the “genetic scissors” from bacteria Streptococcus pyogenes’ CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas immune system. The CRISPR-Cas9 system is able to precisely cut DNA under direction of a sgRNA approximately 100 nucleotides long. The sgRNA is made up of two main sections: the “scaffold” region, also known as “direct repeat region” which provides structure; and the “spacer” region, which is specific to the target DNA sequence. This combination allows for a precise double-strand cut to be made.

Another application of guide RNA-mediated editing is base editing. In base editing, a single base pair is targeted and transformed from C-G to T-A, or A-T to G-C. For editing to occur, guide RNA recruits the modified Cas9 protein and deaminase protein to the target sequence. Base editing allows for targeted editing of the DNA strand, while only having to nick (cut) one strand of DNA.

There are many applications of gene editing. In ex vivo gene editing, cells from a patient are extracted, edited, and then reintroduced to the patient (autologous cell therapy), or similarly using the cells from a healthy donor (allogeneic cell therapy). A challenge to highlight in ex vivo gene editing is that the edited cell must be able to survive the extraction, manipulation, and transplantation process, as well as potentially replicate when returned to the body.

In contrast, in vivo gene editing is used in cases where the patient’s cells cannot be removed and reintroduced, as in the case of some diseases that target the central nervous system and the musculoskeletal system. Ensuring safe and efficient delivery of the essential ingredients for editing the target tissue, not to mention controlled cell editing within the patient, are potential limitations in in vivo gene editing.

The field of gene editing is rapidly diversifying and evolving. CRISPR Car-T cells designed to target cancerous leukemia and lymphoma cells are in clinical trials, as are CRISPR-based treatments of sickle cell disease. The therapeutic potential of guide RNA in developing novel therapies across a broad spectrum of disease states is vast.