Revisiting the Medical Implications of CRISPR-Cas9
AHMAD NISAR – The discovery of the CRISPR-Cas9 construct, like penicillin, was an unexpected product of microbial research. First recognized in E. coli and Haloferax mediterranei, clustered regularly interspaced palindromic repeats (CRISPR) and the Cas9 endonuclease were classified as a prokaryotic defense mechanism perfected over eons of antiviral warfare. Following viral infection, bacteria retain bits of the offending viral genome (‘spacers’) intersped between palindromic repeats that signal polymerases to transcribe these spacer sequences in the event of another viral infection. This spacer-derived RNA (crRNA) binds to a complementary sequence in the invading viral genome with the assistance of tracrRNA, guiding the Cas9 nuclease to perform a ‘double-stranded break’ in the phosphodiester bonds of the DNA helix backbone. Although this ingenious mechanism has been described by various microbiologists since the late 1980s, its application in the field of ‘genome editing’ was not so immediately recognized.
However, in 2012, the labs of Emanuelle Charpentier and Jennifer Doudna developed a chimeric single guide RNA (sgRNA) incorporating both crRNA and tracrRNA, allowing for controlled pairing of the sgRNA construct with Cas9. The sgRNA construct could be adapted to a specific target gene in any given genome. The subsequent double strand breaks inflicted by Cas9 could be repaired through TALEN or ZFN-mediated homologous recombination with a foreign gene of interest. ‘Nicks’ in DNA could even be introduced via this localizing construct with a deaminase altering cytosine to uracil (forcing endogenous DNA repair) coupled with a reverse transcriptase to replace ‘nicked’ DNA. This is done using a newly reverse-transcribed sequence derived from RNA situated within the sgRNA construct. In October 2020, Charpentier and Doudna received the Nobel Prize in Chemistry for their clever streamlining of these processes. A simple — yet highly specific — construct can now be used to routinely delete or introduce sequences of any origin into any organism, including humans.
The ease and efficiency with which CRISPR-Cas9 operates may soon launch it into mainstream use. Just as the now-prolific polymerase chain reaction (PCR) technique has cut the time required for genetic sequencing from weeks to mere hours, CRISPR-Cas9 has pushed the possibility of ‘germline gene editing’ from the pages of sci-fi novels into the labs and workbenches of scientists, medical professionals, governments, and corporations around the world. Spacer-complementary DNA can be introduced into targeted locations of the genome to serve as guides for the sgRNA-Cas9 construct to freely introduce or delete required sequences. This technology poses a number of ethical constraints. Primarily, the notion that germline editing will not be universalizable could amplify and exacerbate existing inequalities. However, the relative ease with which CRISPR is applied makes such ethical postulations seem esoteric. In reality, a multitude of global teams are already racing to further refine and apply the technology to many different organismal systems. Its application into the human genome is thus close to inevitable.
Prior to the streamlining of this process, in 2017, researchers had successfully utilized CRISPR-mediated germline editing to replace mutant embryonic copies of a gene expressing the MYBPC3 protein (a deficiency of which is responsible for hypertrophic cardiomyopathy). It is quite possible that scientists in the near future will be able to replace overactive oncogenes or deficient tumor suppressor genes in embryos to preempt the development of cancer in adulthood. They could even genetically screen embryos to ensure a lack of disruptive congenital disorders. ‘Designer children’ with superhuman intelligence, strength and beauty is, however, a still far-off conception, as these phenotypic traits are not often encoded by discrete genetic units, causing the endeavors of 20th century human eugenics to prove futile. Regardless, the introduction of germline editing technology into medicine will be unavoidable as it will be difficult for many parents to deny their potential children a disease-free life potentiated by embryonic editing. These issues are sure to be confronted by many now entering the medical field.
Sources:
https://www.livescience.com/59971-crispr-used-on-embryos-in-us.html
Copy Editor: Sophia Bartell
Photography Source: Bianca Patel, https://www.statnews.com/2019/04/17/crispr-embryo-editing-ban-opposed-by-families-carrying-inherited-diseases/