The prospect of a cure for genetic disease has had a tremendous impact on the direction of science, but it’s moving ever closer to becoming a reality thanks to the introduction of gene modification. This revolutionary technology known as CRISPR has erupted in popularity, opening up the doors to editing our genes and addressing genetic mutations through the use of specialized enzymes. These CRISPR arrays possess a unique ability to recognize and target specific segments of DNA, enabling a Cas9 enzyme to splice this segment at any targeted location along a gene and completely alter the way this gene is expressed (Baffoe-Bonnie, 2019). Despite the promise that these modification abilities hold for our future, testing for their application on human cells is in an early stage of development, which presents many unknowns as to what gene editing may bring for us in the long-term. With CRISPR being such cutting-edge technology, the research available for understanding its usage outside of a laboratory setting is still quite scarce, leaving plenty of room and a need for researchers to look beyond its general effects before we can truly integrate CRISPR into our daily lives (Perez-Pinera et al. 2012).
Sickle cell disease is just one of the many being looked into for testing purposes, not only for the clear success it has exhibited so far in research but the hope that this brings of nearing a long-awaited cure. Being the most common monogenic disease worldwide, sickle cell disease stems from a point mutation present in the hemoglobin gene, which, when affected, codes for abnormally shaped red blood cells and a shortage of oxygen transport within the body (Voit et al. 2013). This condition has long faced neglect within the world of research, seeing limited recognition of its true severity worldwide and insufficient availability of curative research (Baffoe-Bonnie, 2019). However, by introducing a double-strand break, CRISPR technology is capable of accurately targeting the hemoglobin that codes for sickled cells and making a correction to enable the production of healthy, oxygen-transporting cells in those affected (Hoban et al. 2016). Numerous studies have now been conducted as a means of testing the efficiency and effectiveness of different stem cell modifications as treatment options for SCD, and though these have determined that gene editing is an appropriate treatment, how to actually pursue these treatments remains in question.
One particular study conducted by Megan Hoban, Dianne Lumaquin, and several others at the University of California, Los Angeles, did work to answer this unknown: how do CRISPR and TALEN (transcription activator-like effector nucleases that also function to splice specific sequences of DNA) compare in effectiveness for treating sickle cell disease and which areas of these modification techniques still present the need for rectification. They not only strove to understand if these were plausible correction methods, but how these methods performed comparatively in their target cleavage rates, nuclease specificity, and ability to induce chromosomal rearrangements specifically at the β-globin loci responsible for sickle cell disease (Hoban et al. 2016).
The actual tests were performed through a series of five primary methods, consisting of cell culturing, CD34+ cell processing, electroporation, high throughput sequencing, as well as off-target analysis to determine results (Hoban et al. 2016). While it may sound complicated, the first three steps were simply a way of collecting cell samples that CRISPR and TALEN could perform on by isolation of their β-globin mutation. The acquired blood samples were donated by a number of local sickle cell patients and consisted of K562 and CD34+ cells, cell lines that are both found in human bone marrow and blood which contain the β-globin gene. Once these samples were obtained and their hemoglobin genes were isolated, equimolar amounts of TALEN and CRISPR DNA were added to each and analyzed for their ability to target the hemoglobin genes during electroporation (a process in which a pulse of electricity is applied to open up pores in the cell membrane and allow DNA to be introduced). The optimized efficiency that both were able to locate, cleave, and repair this gene could then be compared based on the presence of an NGG PAM sequence within the DNA of each strand (Hoban et al. 2016).
After comparing the performance of CRISPR and TALEN technologies, the findings of the study showed that CRISPR/Cas9 RNAs were able to produce a rate of 4.2-64.3% gene modification of the β-globin gene compared to a lesser 8.2-26.6% from TALENs, confirming researchers’ intentions to pursue CRISPR RNAs as a form of SCD gene correction (Hoban et al. 2016). These results exhibited a higher overall rate and range of gene cleavage in samples treated with CRISPR gRNAs than those provided by the TALEN pairs. In addition to these on-target cleavage rates, researchers also decided to test nuclease off-target cleavage that was produced in each of the samples. Of the 6 CRISPR guides tested, none showed any off-target disruption, a positive sign when you’re working specifically to cleave one particular gene and not others within the body. TALEN-treated samples, on the other hand, saw cleavage of 11% of off-target alleles, making it once again inferior compared to the results of CRISPR gRNAs (Hoban et al. 2016).
Judging only on this information, it would seem that researchers could easily conclude CRISPR to be the premier option for sickle cell treatment. Nevertheless, further testing brought forth the complication that CRISPR gRNAs also demonstrated significantly high levels of allelic disruption, meaning they introduced unwanted deletions near the cleavage site in addition to deletions that were intentional (Hoban et al. 2016). This is yet another demonstration that research must be an ongoing pursuit, as even with the high therapeutic success rates that are produced through CRISPR, there are plenty of opportunities for this technology to be refined before application in treating real patients. Researchers did come to the final decision that CRISPR techniques were still the most appropriate strategy to pursue in SCD gene therapy, yet undoubtedly one that could use improvements in its safety and reliability. This conclusion ushers us one step forward in narrowing in on an effective sickle cell cure, and the closer we come to determining what gene modification method this may be, the closer we are to reaching areas in medicine that were never before thought possible.
The results that emerged were valuable in understanding where the next steps lie for sickle cell treatment and CRISPR enhancement, but the study itself had areas that could benefit from improvement as well. The study demonstrated that it followed appropriate guidelines and was well conducted, but it also possessed a relatively limited sample size obtained through voluntary donation and not random collection. While this does confirm that informed consent applied here, it also makes the data set at risk for not being an entirely randomized sample and leaves room for other variables to be at play. The authors did disclose that for the most accurate determination of which modification reagent is more effective, other factors should have been analyzed alongside their corrective success. While researchers took the approach of analyzing off-target cleavage rates, they did not provide any specific results on toxicity or other harmful effects that may have arisen from CRISPR or TALEN usage on the cells. This could present difficulties in accurately comparing TALEN and CRISPR for safe use outside of a laboratory and may be one of the explanations as to why researchers had a difficult time conclusively stating which treatment was more optimal.
Regardless of the improvements that could have been made, this study served as progress in the direction of understanding CRISPR’s limitations as well as its applications in the world of genetic disease. We must keep pursuing research and asking follow-up questions, as this is the only way that CRISPR technology will continue to grow and possibly reach a point of human delivery in disease treatment. With the success we’ve already witnessed in CRISPR’s ability to modify existing genes and eliminate mutations, we do have hope for the future of genetic treatment, but it’s pertinent that sickle cell disease receives the attention it’s been missing in this department as well. The experimentation pursued by UCLA’s study is one step on the path to getting here, but it certainly will not be the last you hear of CRISPR.
Hoban MD, Lumaquin D, Kuo CY, Romero Z, Long J, Ho M, Young CS, Mojadidi M, Fitz-Gibbon S, Cooper AR, et al. 2016. CRISPR/Cas9-mediated correction of the sickle mutation in human CD34+ cells. Mol Ther. 24(9):1561–1569. doi:10.1038/mt.2016.148. https://www.sciencedirect.com/science/article/pii/S1525001616453323.
Baffoe-Bonnie MS. 2019. A justice‐based argument for including sickle cell disease in CRISPR/Cas9 clinical research. Bioethics. 33(6):661–668. doi:10.1111/bioe.12589. https://onlinelibrary-wiley-com.libproxy.lib.unc.edu/doi/full/10.1111/bioe.12589
Perez-Pinera P, Ousterout DG, Gersbach CA. 2012. Advances in targeted genome editing. Curr Opin Chem Biol. 16(3–4):268–277. doi:10.1016/j.cbpa.2012.06.007. https://www.sciencedirect.com/science/article/pii/S1367593112000762.
Voit RA, Hendel A, Pruett-Miller SM, Porteus MH. 2014. Nuclease-mediated gene editing by homologous recombination of the human globin locus. Nucleic Acids Res. 42(2):1365–1378. doi:10.1093/nar/gkt947. https://academic.oup.com/nar/article/42/2/1365/1028926.
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