CRISPR-Cas9: The Future of Genome Editing

The world of genetic editing has widened over the past few decades in no small part thanks to the CRISPR/Cas system. CRISPR arrays were first discovered in 1987 by Yoschizumi Ishino in E. coli and, again, independently discovered in 1993 by Francisco Mojica in a microbe that can withstand high salt concentration in ocean water—Holferax mediterranei (Lander, 2016, p. 18). What these two scientists had found in these prokaryotes became known as Clustered Regularly Interspaced Palindromic Repeats (CRISPR): segments of interspaced DNA identical when reading a single strand forward, and its complementary strand backwards, with segments of spacer DNA in between the repeats. The spacer DNA between these palindromes was soon discovered to be foreign—typically viral—DNA that had been cut and pasted into the bacterial genome as a sort of adaptive immune system, allowing the cells to recognize and deactivate these viruses after they had previously encountered these threats (Hille & Charpentier, 2016, p. 1). Since then, scientists such as Jennifer Doudna and Emmanuelle Charpentier, have searched for, and found, ways to adapt and apply this system to edit the human genome. CRISPR and the associated (Cas) proteins are the future of biomedical research and personalized medicine due to their precision genome editing properties. However, it comes with a variety of ethical concerns due to the implications of editing germline cells (eggs and sperm).

Figure 1: Cas9 (green) cutting double-stranded DNA (blue/purple) Complex. (Janet Iwasa. 2019.)

How CRISPR/Cas9 Works

Firstly, it is important to understand what Clustered Regularly Interspaced Short Palindromic Repeats mean and how the system works. DNA is comprised of four base pairs—adenine (A), thymine (T), guanine (G), and cytosine (C)—that make up the entire genome of an organism in two complementary strands with A binding to T and C binding to G. Therefore, an example of a palindromic repeat would be if one strand reads as CTGCAG while the complementary strand sequence would be GACGTC; when read backwards, the complementary strand is the same as the template, just like the word ‘madam’ or ‘civic’. Additionally, “the so-called CRISPR array is preceded by an AT-rich leader sequence and is usually flanked by a set of cas genes encoding the Cas proteins.” (Hille, & Charpentier, 2016, p. 1).

In the CRISPR sequence found in E. coli, these palindromic repeats are interspaced by spacer foreign DNA that matched the sequence of a P1 bacteriophage (a specific virus that only infects bacteria cells); this strain of E. coli happened to be resistant to infection by the P1 bacteriophage (Lander, 2016, p. 20). This led to the idea that bacteria use CRISPR and its associated proteins as an immune system. “In this system, the DNA of the invaded virus or plasmid will be cleaved into a novel spacer and stored in an array in DNA. When the same virus or plasmid invades again, the corresponding invading DNA will be recognized and interfered.” (Cui, Xu, et al., 2018, p. 455).

After infection by a bacteriophage, Cas9 recruits the Cas1 and Cas2 proteins—exonucleases capable of ‘snipping’ DNA or RNA strands—to recognize distinct portions of the foreign DNA known as a protospacer and incorporate it at one end of the CRISPR array in a process known as spacer acquisition (Hille, & Charpentier, 2016, p. 2-3). Within any genome, there exists a sequence known as the protospacer adjacent motif (PAM): a small sequence of base pairs (typically the base pair sequence ‘NGG’ where N is any nucleotide) next to the protospacer that can be recognized by a specific domain of the Cas9 protein (Anders, Niewoehner, et al., 2014, p. 1). This both helps with the protospacer selection and ensures that the Cas proteins do not end up cleaving portions of the bacterial genome that contain the inserted protospacer as the CRISPR array itself does not contain the PAM sequence (Hille, & Charpentier, 2016, p. 2-3; Horvath, & Barrangou, 2010, p. 168).

Figure 2: Cas9 Complex. (© Integrated DNA Technologies, Inc. 2021.)

Upon reinfection with the same bacteriophage, the CRISPR array is transcribed into crRNA, containing the segments of the previously acquired foreign DNA. A second type of RNA is required for the maturation of crRNA; this type of RNA is known as trans-activating CRISPR RNA (tracrRNA). After undergoing several steps of modification by Cas proteins, a “complex consisting of Cas9 and a tracrRNA: crRNA duplex targets and cleaves invading DNA” (Hille, & Charpentier, 2016, p. 2). The crRNA is used as a guide, containing the previously acquired protospacer sequence, to target viral DNA while the tracrRNA helps to stabilize the duplex on the Cas9 nuclease which “introduces double-strand breaks in the target DNA.” (Hille, & Charpentier, 2016, p. 3). Thus, the viral DNA can no longer replicate, and the infection is neutralized.

Medical Uses

Though CRISPR-Cas9 has existed as an immune system in bacteria and other microbes likely for millions of years, scientists have recently discovered and engineered ways to use it in animal cells for biomedical purposes. In these cases, the tracrRNA: crRNA duplex is “engineered as a single guide RNA (sgRNA) that retains two critical features”, one being a sequence that matches a specific target in the host’s DNA and another that binds the sgRNA to Cas9 (Doudna, & Charpentier, 2014, p. 1). This allows scientists to target nearly any DNA sequence with Cas9 and a sgRNA, as long as it is adjacent to a PAM; being only a few base pairs long, PAM sequences exist in all genomes (Anders, Niewoehner, et al., 2014, p. 1). Cas9 in conjunction with the sgRNA will unzip and bind to the corresponding target DNA adjacent to the PAM due to the domain of Cas9 that recognizes and binds to PAMs. Once bound to the target site, Cas9 introduces a double-strand break in the DNA at the desired locus sequence (Hille, & Charpentier, 2016, p. 6).

Although the CRISPR acronym has attracted media attention and is widely used in the scientific and popular literature, nearly all genome editing applications are based on the use of the protein Cas9 together with suitable sgRNAs. (Doudna, & Charpentier, 2014, p. 3).

Figure 3: Non-homologous End Joining (left) and Homology-directed Repair (right). (Javier Zarracina. © Vox Media, LLC. 2017. )

This break in the genetic material at a specific point is repaired by the host cell’s genetic repair mechanism through two different processes: non-homologous end joining (NHEJ), or homology-directed repair (HDR) (Hille, & Charpentier, 2016, p. 6). NHEJ is the favored method when the desired effect is to disable the target gene entirely as it typically results in short, random insertions or deletions where the break occurs. Meanwhile, HDR is the favored method when a genetic correction is preferred; during HDR, a segment of DNA that is homologous to (complementary to) each end of the double-stranded breaks is introduced in the cell, and the host’s DNA repair mechanism inserts the segments via homologous recombination to the desired site (Hille, & Charpentier, 2016, p. 6).

Theoretically, the HDR method can be used to correct any number of faulty genes that cause diseases such as cystic fibrosis (CF) which is “a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene.” (Fan, Perisse, et al., 2018, p 1). Using stem cells isolated from two cystic fibrosis patients, the mutation in the CTFR gene was corrected using the CRISPR/Cas9 HDR with the wild-type gene, resulting in transcription of the functional transmembrane receptor that is faulty in CF patients (Schwank, Koo, et al., 2013 p. 653). Though this experiment was confined to human stem cells, the technology has been taken beyond the theoretical in 2018 when the world’s first genome-edited babies were born, raising an array of ethical concerns.

Ethical Implications

In 2018, He Jiankui of the Southern University of Science and Technology in Shenzhen, China used CRISPR/Cas9 to genetically engineer human embryos to be resistant to HIV infection, resulting in the birth of two genetically altered twin girls (Lovell-Badge, 2019, p. 1; Sand, Bredenoord, et al., 2019, p. 1). These twins were born into the world at a time when altering the human germline was not considered safe or ethical, as this technology could alter the genetic makeup of generations yet to come. It even triggered global calls for a moratorium on germline editing (Lander, Baylis, et al., 2019, p. 165). While new and largely untested medical and clinical trials often have detrimental side effects that must be considered when it comes to CRISPR/Cas9, the largest societal concern revolves around the potential for eugenics to be applied through this technology.

Figure 4: He Jiankui. (Anthony Kwan. 2018.)

Another risk, shared globally, is posed by the greater society. It is possible, for instance, that allowing CRISPR germline editing, even if only for medical purposes, might in some respect(s) lead to the return of eugenics, whose proponents believed that the human population can be improved by controlled breeding to increase the occurrence of “desirable”, heritable characteristics. (Brokowski, & Adli, 2019, p. 8).

Throughout history, the selective breeding of people with supposedly desired genes has occurred while the selective ending of bloodlines with undesirable genes through atrocities such as sterilization or outright murder, and these practices continue to be suggested and carried out by a variety of political regimes across the globe (Brokowski & Adli, 2019, p. 8). With the technology available to ostensibly remove these ‘undesired’ genes in eggs and sperm cells, those genes would not only be removed from the embryo—and therefore, eventually, the person born from that embryo—but from the offspring that person may produce, ensuring the continuation of the ‘good’ genes for years to come. Simply put, “eugenics lurks in the shadow of CRISPR. Any opening to the germline, sperm, and egg modification is, simply put, the opening of a return to the agenda of eugenics: the positive selection of “good” versions of the human genome and the weeding out of “bad” versions, not just for the health of an individual, but for the future of the species.” (Brokowski, Pollack, et al., 2015, p. 273-274).

Figure 5: Opportunities and Challenges of CRISPR/Cas9. (U.S. Government Accountability Office. 2020.)

The applications of CRISPR/Cas9 seem to be limitless, from a potentially curative treatment for CF to creating families of livestock resistant to certain devastating diseases. The system’s ability to target nearly any specific gene and alter or delete it altogether is what makes it so unique: its precision engineering abilities. However, that is also what makes it so dangerous. The ability to select individuals for certain genes deemed to be desirable by whoever decides to engineer so-called ‘CRISPR babies' next could lead to the next wave of eugenics-inspired experiments. This amazing technology which has a great capacity for healing, may also result in a great deal of harm.

Bibliographical References

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Visual Sources

Cover Image: Bonazzi, D. (2017) Genes Illustration. [Illustration]. Image retrieved from: https://www.theispot.com/stock/image_popup/id/a3864ir1029

Figure 1: Iwasa, J. (2019). Cas9 Protein Involved in the CRISPR Gene-editing Technology. [Illustration]. The Innovative Genomics Institute at UC Berkley. Image retrieved from https://images.nigms.nih.gov/pages/DetailPage.aspx?imageid2=5816

Figure 2: PAM Site and Cas9 gRNA. ©Integrated DNA Technologies, Inc. (2021). [Illustration]. Image retrieved from https://www.idtdna.com/pages/support/faqs/what-is-a-pam-sequence-and-where-is-it-located

Figure 3: Zarracina, J. (2017). Non-homologous End Joining, and Homology-directed Repair. [Illustration]. © Vox Media, LLC. Image retrieved from https://www.vox.com/science-and-health/2017/8/2/16083300/crispr-heart-disease

Figure 4: Kwan, A. (2018). He Jiankui. [Photograph]. Getty Images. Image retrieved from https://www.scientificamerican.com/article/what-crispr-baby-prison-sentences-mean-for-research/

Figure 5: Opportunities and Challenges of CRISPR/Cas9. U.S. Government Accountability Office. (2020). [Illustration]. Image retrieved from https://www.gao.gov/products/gao-20-478sp