Cautiously Optimistic: CRISPR Technology
By Jenab Diallo
Introduction
In recent years, CRISPR has become one of the most prominent and generally debated scientific topics in the overall public sphere. In many of these spaces, CRISPR, which is short for CRISPR-Cas9, is mainly discussed in terms of the ethical issues surrounding using the technology to edit genes. However, there is an almost 20-year backstory to the development of CRISPR as a technology. This history, what exactly CRISPR is, and its wide variety of uses are not as widely discussed in the public sphere, and yet, they can help to provide a good perspective in considering ethical issues such as genetic enhancements and our significant history with eugenics.
About CRISPR
CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeats, is a natural component of bacterial (prokaryotic organisms) immune systems. The system includes a CRISPR-associated (CAS) nuclease, an enzyme that binds and cuts DNA, and a guiding RNA sequence (gRNA) that directs the CAS nuclease to its target. When invading bacteria, aka bacteriophages, enter, the system cuts the DNA of the virus and disables it. A bit of the virus’s DNA is stored in the bacteria’s genome so that it can be easily used the next time the virus invades. These viral fragment spacers are placed between palindromic sequences, which are sequences where the complementary strands of DNA read the same in both directions. This placement is where CRISPR gets its name. When the virus re-enters the bacterium, Cas9 checks the viral DNA for the protospacer adjacent motif (PAM) which is a short sequence downstream of the target site, then moves upstream looking for the target sequence. This target sequence is provided to Cas9 by guide RNA (gRNA) a complex formed by CRISPR RNA (crRNA), which is complementary to the viral spacer fragment formed and stored in the bacterium’s genome when the virus first attacked, and trans-activating CRISPR RNA (tracrRNA), which acts as scaffolding. The gRNA guides the scissors, in this case, Cas9, to where they need to cut. The Cas9 destroys the virus by creating a double-stranded break which the virus can not fix due to its lack of DNA repair mechanisms.
History
This natural mechanism’s potential for gene editing was first uncovered by Dr. Jennifer Doudna and Dr. Emmanuel Charpentier. They speculated that if they were able to provide a different guide for the Cas9 they could perhaps create cuts at any location in the genome of an organism. Then, because the cut wasn’t on viral DNA and cellular DNA repair pathways were present, these could be exploited to edit the genome (i.e insert new genes/fragments or turn off certain genes). Their discovery, which was published in 2012, was extremely key to the application of CRISPR. This natural mechanism and its potential for gene editing were first uncovered by Dr. Jennifer Doudna and Dr.Emmanuel Charpentier. They speculated that if they were able to provide a different guide for the Cas9 they could perhaps create cuts at any location in the genome of an organism. Then, because the cut wasn’t on viral DNA and cellular DNA repair pathways were present, these could be exploited to edit the genome (i.e insert new genes/fragments or turn off certain genes). Their discovery, which was published in 2012, was extremely key to the application of CRISPR, however, the history of CRISPR goes back almost 20 years.
As far as we know, it started with Francisco Mojica who began his doctoral studies at the University of Alicante and joined a lab working on Haloferax mediterranei: an archaeal microbe that has extreme salt tolerance. While analyzing the DNA fragments of this genome, he noticed repeated sequences of 30 bases that were palindromic and separated by spacers of 36 bases. He published this finding in 1993. Then, he found similar structures in related halophilic archaea and other distant microbes. Eventually, after leaving for a postdoctoral job at Oxford then returning, Mojica went on to continue to study these repeat structures and named them Short Regularly Spaced Repeats (SRSRs) which he then suggested be changed to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). At this time and until 2003, Mojica found CRISPR loci in 20 different kinds of microbes but nothing was known about their functions. In 2003, Mojica shifted his focus from the repeats to the spacers and he went on to extract each spacer and insert it into the BLAST program to search for similarities with other DNA sequences. He found that one of the spacers from the CRISPR sequence he had gotten from E. coli matched with the sequence of a P1 phage that often infected E.coli strains. After running more of these spacers, he would conclude that CRISPR must play an important role in the adaptive immune system of microbes. It took him almost two years, until 2005, to get these findings published.
Many scientists and studies followed. In 1997, human geneticist Gilles Vergnaud was asked by the French Ministry of Defense to study forensic microbiology. This was in response to reports that Saddam Hussein and the Iraq regime were working to develop biological weapons. The Ministry of Defence wanted to develop methods to trace the source of pathogens based on subtle genetic differences among strains. Vergnaud was working on using tandem repeat polymorphisms, such as what is used in forensic DNA fingerprinting, to characterize different strains of bacteria responsible for anthrax and plague. While looking at samples from a plague outbreak in Vietnam in 1964–1966, he found that closely related 61. Y pestis isolates were identical at their tandem repeat loci except for what his colleague Christine Pourcel identified as CRISPR sites. The strains would have new spacers acquired at the front end of the CRISPR locus. They then found that the spacers corresponded with prophages present in the Y.pestis genome. Thus, it was proposed that the CRISPR locus serves as a defense mechanism and that it holds “the memory of past aggressors.” Meanwhile, a third researcher, Alexander Bolotin submitted a paper just after Mojica’s 2005 one that proposed that transcripts of the CRISPR locus worked by antisense RNA inhibition of phage gene expression. As we know, from the mechanism now, this was wrong. Then, in 2007, Phillipe Horvath along with Rodolphe Barrangou and Sylvian Moineau became the first to successfully experimentally demonstrate Mojica’s theory using a well-characterized phage-sensitive strain S.thermophillus and two bacteriophages. They observed acquired immunity taking place and found that immunity depended on a precise DNA sequence match between spacer and target. Sylvian Moineau would then follow up on this with colleagues. In 2008, while studying plasmid interference in S.thermophilus, they discovered that Cas9 cuts DNA precisely at locations encoded by crRNAS. The role of crRNAs was already studied by John van der Oost and the role of tracrRNA would be studied by Emmanuelle Charpentier and Jörg Vogel.
Many other pieces would come to fall in place. There were still a few key significant contributions to the study of CRISPR and the development of CRISPR technology. Feng Zhang developed and initiated the use of CRISPR in eukaryotic cells and discovered new Cas variants. George Church was among the first to demonstrate CRISPR use in human cells. And finally, biochemist Virginijus Siksnys discovered the ability of CRISPR genes to edit genes in other organisms. This complex history makes clear that the enormous concept of CRISPR, so generally discussed right now, has been built on years of research. Every aspect of the mechanism previously mentioned in this paper took years to uncover and we have now reached a point where this work has produced a powerful tool.
Methods + Ethics
Various methods make CRISPR a powerful tool for genome editing that can be used in a range of ways. As was previously mentioned, because in CRISPR-Cas9 genome editing Cas9 is not cutting into viral DNA but instead into DNA where cellular DNA repair pathways are present, these pathways can be exploited. For example, in the case that Cas9 cuts the DNA and the repair path is non-homologous end joining (NHEJ), the gene becomes non-functional. This is because NHEJ usually results in insertions and deletions in the region being repaired. These can result in a frameshift mutation if they are within a coding region of the gene. This method is known as CRISPR gene knockout and is used in many areas of study like disease modeling, drug discovery and screening, pathway analysis, and functional genomics. On the opposite end is the CRISPR knock-in method where repair takes place via homology-directed repair (HDR) and researchers can insert a new piece of DNA or an entire gene. Knock-ins are much harder to do than knock-outs. However, like knock-outs, they are useful particularly in increasing the viability of immortalized cell lines, precision disease modeling, and production of recombinant proteins. There are also methods for CRISPR activation and upregulation of gene expression (CRISPRa) and interference and downregulation of gene expression (CRISPRi). These methods have been particularly applicable in developmental biology, infectious disease, disease progression, functional genomics, and screening for genetic elements that mediate drug resistance. Finally, most recently base and prime editing have also come into play. These CRISPR methods do not use Cas9 and instead use catalytically dead Cas9 or nickase Cas9. They are far more precise and induce single nucleotide substitutions.
The applications of CRISPR technology are already broad and continue to grow. Perhaps the most commonly known is CRISPRs role in medicine, particularly when thinking about gene and cell therapies that could potentially cure many genetic diseases. The first trial of this kind of therapy was performed in 2019 treating patients with sickle cell disease. The treatment worked and clinical trials involving other genetic diseases continue to take place. Another application of CRISPR technology is in diagnostics. It is currently being used in test kits for COVID. In agriculture, researchers are working on using CRISPR to create crops that are disease and drought-resistant. And finally, as we work to move away from fossil fuels, CRISPR is being used to study and advance the use of biofuels.
While the applications of CRISPR are broad and will continue to grow, the main focus of most ethical debates has to do with the technology’s role in human genome editing/gene therapies. The concept of gene editing is not particularly new. However, CRISPR technology poses a particular situation where gene editing could become far easier. Furthermore, in cases where the human germline is edited, these changes will be passed onto future generations. Both of these factors present questions of safety, consent, and equity.
Despite all of the research done thus far, there is still a lot we don’t know about CRISPR and what the long-term effects of using it for genome editing could be. More research is needed, however, there are cases where further research is less likely because there are key religious and moral objections to using humans and human embryos. While in some cases gene therapy and editing may be necessary (i.e two prospective parents homozygous for a disease) there are many worries that the use of genomic editing to handle these cases and treat diseases is a slippery slope toward using CRISPR for genetic enhancements. Another topic of debate is informed consent. There is no way to receive informed consent from those impacted by genome editing because they include embryos and future generations. Last, but certainly not least, there is concern about equality as we move forward with CRISPR. Many are worried that genome editing will only be accessible to the wealthy thus increasing health care disparities that already exist. This accessibility issue along with the ability to pick and choose specific genes over others brings up a familiar issue- eugenics.
Conclusion
I share concerns about every one of these ethical issues. However, after countless years of research and development of methods and applications for CRISPR, I believe it is important to follow through. By following through, I mean to follow through in studying the technology and applying it to the issues we face such as diseases and climate change (biofuels and agriculture). However, as they say, with great power comes great responsibility. CRISPR is a powerful tool we have that can be used to do amazing things and change lives for the better. At the same time, we now have an even greater responsibility to ensure that this technology helps to bridge the gaps rather than widen them. It is our responsibility to ensure that we properly regulate the use of CRISPR towards needs rather than wants. Finding that line will be challenging but beyond important.
