By Tershona Branch
The day has come- more importantly, the technology has arrived. For thousands of years, disease was a death sentence. If a child was born deformed, it was looked at as a curse and the child was ostracized. If a child was born with a genetic disease, such as cystic fibrosis, the child had a low life expectancy under 10 years of age. However, in the past two centuries, we have given children a few more years through advancements which prepare families for children with genetic disorders. Some of these diseased children have a greater life expectancy as a result of developments in medicine. These individuals with genetic disorders were given options to manage their disorders, live to the age of reproductions, and have families. Today, we have new technology that can edit genes at the moment of life, giving children the opportunity to live normal, healthy, disease-free lives. This new gene editing technology is called CRISPR. CRISPR/Cas9 is a gene-editing technology that has effectively repaired genetic disorders in mice, modified human embryos, and has clinical potential. In the future, after many trials and lots of research, CRISPR could be used to treat genetic disorders during gestation, over the course of a lifetime, and after they may be acquired. The various ways that CRISPR technology can be used will change healthcare and the way we approach inherited diseases.
CRISPR, standing for Clustered Regularly Interspaced Short Palindromic Repeats, has a history stretching thirty-one years of constant research. Basically, it is a series of DNA segments from a prokaryote with base sequences that repeat. The segments of DNA are separated by “junk” DNA. Accidentally, like most things in science, CRISPR was discovered in 1987 when scientists at Osaka University cloned part of CRISPR with another gene; however, they did not understand its significance. In 1993, 2000, and 2003, scientists researched CRISPR, finding its origins in archaea and began understanding it better. However, it wasn’t until 2005 that Cas9 was discovered; ten years later, scientists discovered that CRISPR/Cas9 makes effective gene edits in human zygotes (CRISPR TIMELINE).
CRISPR consists of an RNA guide that searches an organism’s genome for the target gene and Cas9 that allows the modification to be made. CRISPR scans the genome for a specific sequence of amino acids. Many CRISPR sequences recognize over twenty nucleotides that are very unique to a species, allowing for much specificity (Zhang et al. 2014). Activation of Cas9 activates the cleavage process, cutting both strands of the organism’s DNA at the desired location (Redman et al. 2016). Changes can be made as specific as one single base pair with the Cas9 protein. Essentially, CRISPR/Cas9 disrupts the genome, deletes a segment of the DNA, and corrects the DNA; other proteins come behind CRISPR/Cas9 to seal and activate the DNA. Proteins, amino acids, hormones, and other biological operations shift away from undesired diseases. Today, there are three different methods that CRISPR can be used: ex vivo, in vivo, and in vitro fertilization.
Ex vivo treatment, meaning “out of the living” in English, extracts cells from the patient then treats the cells in a culture. CRISPR/Cas9 acts on these cells within the culture. Today, scientists are able to evaluate and select the corrected cells to put back into the patient- instead of putting all the cells back in the patient (Savic and Schwank 2015). As a result of the ability to properly evaluate these cells, concerns of accuracy are less pressing in ex vivo therapy as compared to in vivo therapy. In 2007, Takahashi successfully reprogrammed human somatic cells providing a gateway to clinical treatment. One of the first diseases that ex vivo CRISPR was used to combat was b-thalassemia, a genetic disorder that reduces the production of hemoglobin, resulting in a lack of oxygen in most of the body. Through combining the Cas9 technology with the piggyBac sequence that replaces the mutation, scientists successfully corrected both mutations causing b-thalassemia disorder (Xie et al. 2018). Secondly, ex vivo CRISPR technology was used to treat somatic stem cells to treat cystic fibrosis-causing allele. Cystic fibrosis is a genetic disorder affecting the lungs, causing difficulty breathing, frequent lung infections, and infertility in males. It results from a mutation in the CFTR, cystic fibrosis transmembrane conductance regulator, leading to accumulation of mucus in gastrointestinal and pulmonary tracts. First, CRISPR/Cas9 was targeted at adult intestinal stem cells. Within two weeks, a single cell generated organoids, confirming CRISPR/Cas9 has the potential of editing adult human stem cells, specifically intestinal organoids. When CRISPR was further tested in adult stem cells with a focus on CFTR, results indicated high specificity in adult human stem cells (Schwank et al. 2013). Today, scientists are using ex vivo CRISPR/Cas9 based therapy to approach HIV. Approaches include eliminating integrated HIV DNA from T cells and imitating a resistance to HIV virus through destroying a receptor protein needed for HIV T cell infection. Ex vivo alone has been used to find approaches to cure life-threatening diseases that plague and to decrease the life expectancy of many around the world.
In contrast to ex vivo, in vivo means “within the living.” CRISPR/Cas9 is delivered as a therapeutic biological package, such as a lipid, to specific target organs. The first successful in vivo CRISPR/Ca9 corrected genetic disease was in a mouse by Yin et al. for tyrosinemia (2014). Tyrosinemia is a genetic disorder that results from a break down in amino acid tyrosine, leading to its build up in the body. Intravenous injection of vectors with Cas9 and new DNA delivered the therapeutic CRISPR technology directly to the mouse’s liver. This study was successful and, more importantly, indicated that a true chance of genetic correction with CRISPR/Cas9 in humans (Yin et al. 2014). Other studies have shown that CRISPR/Cas9 can be used in humans to cure hepatitis B virus infection. Despite this true success, research is still needed for in vivo therapy because there may be errors at zero to 150 different sites.
In the future, in vitro fertilization-mediated-CRISPR may be the approach most preferred. In vitro fertilization (IVF) is artificial conception that gives many couples a better chance of conceiving than through natural fertilization. There are two ways that CRISPR is used in IVF: treating the zygote by injecting cells with CRISPR after fertilization and injecting sperm and CRISPR into the egg at the same time. With fertilization then injection, not all of the embryos were successfully edited (Bevacqua 2016). However, when sperm and CRISPR were injected together, scientists find better results. Significantly less errors appear in embryos with simultaneous injection (Connor 2017). IVF treatment allows editing of the genome before most of the cells have developed which can prove to be less straining on a patient and improve quality of life.
CRISPR possesses unlimited possibilities of changing humanity’s approach to genetic diseases. This limitless possibility is concerning for many people. Some people think that eugenics and cosmetics may become the main concern of this technology instead of health care. For this technology to truly thrive for the best purposes, United Nations or individual nations of the world need to work with scientists to work out guidelines that must be followed and enforced to ensure proper and appropriate use. CRISPR is not just a mystery and jewel of science, but one of health and social justice. This technology can change the world one embryo or treatment at a time, but the only way to know if we are truly prepared for this drastic change is to have proper legislation.
References
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Connor S. 2017. First human embryo edited in U.S. [Internet]. MIT Technology Review; [cited 2018 Sept 10]. Available from https://www.technologyreview.com/s/608350/first-human-embryos-edited-in-us/.
CRISPR TIMELINE [Internet]. Available from https://www.broadinstitute.org/what-broad/areas-focus/project-spotlight/crispr-timeline
Redman M, King A, Watson C, King D. 2016. What is CRISPR/Cas9? ADC Education & Practice. 101(4):213-215.
Savic N, Schwank G. 2015. Advances in therapeutic CRISPR/Cas9 genome editing. ScienceDirect. 168:15-21.
Schwank G, Koo B, Sasselli V, Dekkers J, Heo I, Demircan T, Sasaki N, Boymans S, Cuppen E, van der Ent C, Nieuwenhuis E, Beekman J, Clevers H. 2013. Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell. 13(6):653-658.
Xie F, Ye L, Chang J, Beyer A, Wang J, Muench M, Kan Y. 2014. Seamless gene correction of b-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac. Genome Research. 24:1526-1533.
Yin H, Xue W, Chhen S, Bogorad R, Benedetti E, Grompe M, Koteliansky V, Sharp P, Jacks T, Anderson D. 2014. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. nature biotechnology. 32(6):551-554.
Zhang F, Wen Y, Guo X. 2014. CRISPR/Cas9 for genome editing; progress, implications and challenges. Oxford Academia. 23(R1):R40-R46.