Hailed as one of the greatest scientific breakthroughs of the 21st century—a revolutionary tool for bioengineering with civilization-altering implications for the future of medicine, agriculture, and civilization itself—a seemingly innocuous little enzyme by the name of CRISPR Cas-9.
This technology is far from a synthetic invention, cooked up in simmering sickly-green vials in fluorescent-lit laboratories by geneticists drunk on their own power—the reality of its discovery is far more miraculous.
The first breakthroughs were incidental: Japanese molecular biologist Yoshizumi Ishino at the Research Institute for Microbial Diseases in Osaka was attempting to characterize isozymes (enzymes with different polypeptide configurations but identical biological functions) of alkaline phosphatase (an enzyme responsible for molecule dephosphorylation) in various strains of E. coli. He published his results in 1987, concluding that isozyme formation was catalyzed by the proteolytic cleavage of terminal arginines post-translation, mediated by the iap gene—first sequenced by Ishino and his colleagues. In doing so, he stumbled upon a peculiar series of repetitive nucleotide sequences flanking iap.
The paper reads, “An unusual structure was found in the 3'-end flanking
region of iap (Fig. 5). Five highly homologous sequences of 29 nucleotides were arranged as direct repeats with 32 nucleotides as spacing” (5432, Ishino et al).
These so-called clustered regularly interspaced palindromic repeats—CRISPR, for short—were noted as nothing more than a scientific oddity at the time. Operating with limited, costly, and cumbersome manual sequencing technology, Ishino and his team conclude, “So far, no sequence homologous to these has been found elsewhere in prokaryotes, and the biological significance of these sequences is not known.”
In following years, variability in CRISPR sequences were used merely as an identification guide for different strains of pathogenic bacteria. It wasn’t until 1995 that the sequences were given newfound scientific weight: Francisco Mojica of the University of Alicante, Spain discovered similar sequences in the genomes of archaea — primitive single cellular organisms evolutionarily and geologically distinct from bacteria. The convergent evolution of these sequences indicated their role of great biological importance, which was determined to be the identification of specific DNA sequences. Mojica compared the snippets of codons contained within this molecular catalog and noted that they matched fragments of bacteriophage DNA, indicating that CRISPR functions as a primitive sort of immune system, allowing prokaryotes to identify foreign genetic information. Subsequently, researchers at the French National Institute for Agricultural Research discovered that all CRISPR genes are flanked by cas genes encoding for the production of large proteins theoretically capable of cleaving nucleic acids—what would later be christened cas9; the functional enzyme in the CRISPR-cas9 complex. CRISPR genes are also universally accompanied by protospacer adjacent motifs (PAM) sequences critical for site recognition, functioning as a marker to guide cas9 activity. In 2007, French researcher Philippe Horvath experimentally proved that S. thermophilus bacteria are capable of integrating new bacteriophage genomes into their CRISPR sequences, definitively proving its function as a prokaryotic adaptive immune system, with CRISPR sequences in place of antigen presenting cells and cas9 in place of cytotoxic (killer) T cells. Subsequent discoveries unearthed small fragments of CRISPR-associated RNA (crRNA) transcribed from segments of the CRISPR gene locus, seemingly responsible for guiding cellular proteins to foreign viral genetic material.
The critical breakthroughs in this technology, however, occurred in 2011 at the University of Vienna, when a research team headed by Emmanuel Charpentier discovered the final piece of the puzzle: tracrRNA, a small segment that forms a compound complex with crRNA and directs the cas9 enzyme to DNA sequences matching the genetic “profiles” contained within the CRISPR locus, facilitating their cleavage and destruction.
Charpentier joined ranks with biochemist Jennifer Doudna in 2012, publishing a landmark paper that compiled all the fragmentary research regarding the CRISPR cas9 complex into one conclusory declaration of stunning significance: this gene/RNA complex has the capacity to integrate specific pathogenic codon sequences from prior viral attacks into the CRISPR locus; direct cas9 to the target genetic material with the aid of guideRNA; and then cleave the foreign sequence: a pair of molecular scissors of extraordinary precision capable of editing DNA. By bypassing the CRISPR catalog of pathogenic IDs and designing specific RNA sequences to “feed” into cas9, Doudna and her team of researchers succeeded in guiding the enzyme to target and cleave specific DNA sequences, even when isolated from the biological context of prokaryotic immunity in which it originated. CRISPR cas9, already capable of inducing double-strand breaks in DNA to destroy specific sequences, could subsequently be paired with homologous recombination (a cellular process for repairing DNA involving the use of an intact genetic “template”) to zero in on a specific loci on a chromosome and edit, delete, or insert any desired genetic sequence. The implications are eye-poppingly delightful.
What was created was a novel system of genetic engineering of remarkable efficiency, precision, and simplicity: the gateway to a new frontier of genomic modification. Vastly cheaper and more streamlined than older, more cumbersome technologies, CRISPR cas9 revolutionized industries spanning the gamut from agricultural science to oncology. Already, CRISPR has been utilized ed to genetically enhance nutrient-poor rice crops for excess carotenoid production to prevent vitamin deficiencies in developing countries; repair the point mutation in hemoglobin responsible for sickle cell anemia; and excise plasmids responsible for antibiotic resistance in pathogenic “superbugs,” re-sensitizing them to antibiotic therapies.
The future of this technology is simultaneously radiating with revolutionary scientific potential and fraught with ethical controversy—the increasing accessibility of such a highly potent tool for genomic engineering to governments and corporate leaders rightly raises serious concerns about its future applications. The likely development of eugenics-adjacent movements to create “designer babies;” the exploitation of CRISPR to manufacture pesticide-resistant crop cultivars, enabling the use of highly corrosive and toxic chemical concoctions by agricultural monopolies [Monsanto]; and attempts to control quote-on-quote “pest” populations of wild animals through induced infertility (to disastrous ecological effect) are all forebodingly realistic propabilities in the coming years and decades. CRISPR holds both tremendous promise and peril: enabling humanity to tinker with the most fundamental molecular infrastructure of life itself. Only time will tell if we will succumb to the sinister siren song of private profiteering—allowing this miraculous biomedical technology to be weaponized for self-interested ends—or bear this unprecedented power and responsibility with appropriate gravitas and discernment, employing it in our pursuit of a healthier, happier, more prosperous society.
[1] Bolotin, Alexander, et al. “Clustered Regularly Interspaced Short Palindrome Repeats (CRISPRs) Have Spacers of Extrachromosomal Origin.” Microbiology, vol. 151, no. 8, 1 Aug. 2005, pp. 2551–2561, https://doi.org/10.1099/mic.0.28048-0.
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