Functional genomics is the discipline that investigates function of genes through use of synthetic oligonucleotides or longer nucleic acids in living cells in ways that alter the genome or changes gene expression. Tools include antisense oligonucleotides (ASOs), RNA interference, splice-switching oligonucleotides (SSOs), and genome editing. Even though genome editing methods have existed for years, it has caught the trend recently due to the emergence of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR). The traditional methods of genome editing include zinc-finger nucleases, TALENs, and megaTALS. These older technologies required protein engineering for every different target and scientists had to create a new nuclease each time they changed the target site, which could take six months of work. But in CRISPR, the protein component stays the same and only the guide RNA (gRNA) changes, which can be mass produced. Consequently, thousands of sites can be simultaneously monitored and changed as desired. This new method has considerably reduced the inconvenience and the time lag associated with site changes. The barrier to working with genome editing technology has been vastly reduced and has opened up new possibilities towards correcting human ailments through gene therapy.
A genome editing experiment with CRISPR requires the presence of both the Cas9 nuclease and a gRNA in the cell. In bacteria, where CRISPR naturally occurs, the gRNA is comprised of two separate RNAs that anneal to form an active complex, a short crRNA (that contains a 20 base target-specific domain) and a longer tracrRNA (that directs binding to Cas9). Alternatively, the gRNA can be artificially reduced to a single RNA species by fusing the crRNA and tracrRNA together into a single-guide, or sgRNA (this version is easier to express from artificial DNA constructs). In practice, genome editing can be achieved by two different approaches. One method is to insert the coding sequences for the Cas9 nuclease and a sgRNA into a plasmid or viral expression vector, introduce this into a cell, and have the cell itself make the gene editing machinery. A second method is to employ recombinant Cas9 protein in a “DNA-free” approach. Here synthetic gRNA (crRNA+tracrRNA pair or sgRNA) is bound to the Cas9 protein in vitro and the resulting ribonucleoprotein (RNP) complex is introduced into cells. Once the Cas9/gRNA complex is in the cell, cleavage of genomic DNA will occur leading to a double-strand break. This break is healed by repair machinery and can lead to changes in the DNA sequence that may disrupt gene function (via non-homologous end joining, or NHEJ) or alternatively can be directed to precisely alter sequence in a desired way (using homology directed repair, or HDR).
“Though it can take a long time for new technologies to create useful drugs, do not give up the quest—keep working on it”
Though used extensively, the plasmid-expression approach can lead to unwanted side effects. For example, the plasmid itself can get integrated into the cell’s genome. Also, the sustained high levels of expression of Cas9 and the gRNA that results from plasmid vectors can lead to cleavage at unintended sites in the genome (off-target effects, or OTEs). The OTE problem is similar to mis-priming in PCR, where the wrong DNA sequence is amplified. In CRISPR, the wrong sequence site is cleaved–obviously something that is not desired. In contrast, the RNP approach provides a “fast on/fast off” process that has high efficiency with reduced side effects. In spite of the lower OTEs seen using the RNP method, unwanted cleavage events still can occur and are a particular concern for medical applications.
Several groups have reported properties of mutant Cas9 nucleases that show reduced OTEs, however these mutants also have reduced on-target activity and are most useful using plasmid overexpression methods; the reduced activity presents problems when using RNP methods. At Integrated DNA Technologies’ (IDT), our team recently generated a novel Cas9 mutant that has both higher specificity and retains sufficient on-target activity to be used in RNP methods. This new mutant is already finding wide utility systems where precision genome editing is required, including making disease models systems for research, stem cell manipulation, and medical applications.
Prospective Use of CRISPR in Various Field Studies
One medical application of CRISPR genome editing technology under intense investigation today is to correct gene mutations in patient-derived stem cells outside of the body (ex vivo methods) and re-infuse the “fixed” cells back into the same patient. This is particularly attractive to treat various hemoglobinopathies such as sickle cell anemia or beta-thalassemia, where CD34+ bone marrow stem cells can be easily obtained and modified in a medical laboratory. Apart from that, the liver has a great prospect in gene editing as scientists have already developed advanced delivery technologies to transport plasmids or large molecular drugs into this organ.
There is a grand future for nucleic acids in medicine, both in therapeutics (as discussed above) and in diagnostics. For example, DNA testing is now commonplace to characterize genetic disorders or to identify infectious diseases through their nucleic acid signatures, such as viral infections or drug resistant bacteria. Cancer treatment is being revolutionized by the ability to identify tumor-specific mutations in key genes as a guide to rational selection of therapy. Every new technology that emerges leads to excitement about potential revolutionary new medical treatments, however it usually takes many years of R&D before widespread adoption occurs. It took many years between the discovery of monoclonal antibodies or antisense technology before the first FDA-approved therapies made it to market. Likewise, it will take time before genome editing therapies attain widespread medical use. If the past is any prediction of the future, problems will be found and these problems will be solved, so long as researchers “stay in it for the long haul”.