Nature’s molecular scissors in the treatment of disease.
Writer: Shivaani Iyer
Editor: Katie Dale
Artist: Lucie Gourmet
‘Revolutionary’ is a term that has crept its way into modern science and medicine. The birth of scientific revolution as a concept can be traced to philosopher Thomas Kuhn, who put forth his understanding of ‘revolutionary science’ as an outcome of crisis, through which a discovery emerges almost as a phoenix from the ashes.
Breakthroughs in biology, however, rarely fit the parameters of his definition. Instead, it can be argued that a discovery can only truly be identified as revolutionary when it propagates into a range of different applications. The recent Nobel Prize nomination to Emmanuelle Charpentier and Jennifer Doudna has drawn attention to the trajectory that CRISPR technology has taken into agriculture, transport, and the most striking yet, medicine. And so, we are naturally drawn to the question: what is CRISPR?
A DNA sequence found in the genome of E. coli shattered the illusion of genome editing as merely a futuristic concept. CRISPR is a bacterial guide RNA (gRNA) component of the prokaryotic adaptive immune system that can recognise viral DNA. When complexed with Cas proteins, it guides the sequence-specific cutting of double stranded DNA to protect against viral infection. In genetic engineering, we can design a gRNA to recognise a specific sequence within the human genome, to direct the Cas nuclease to generate a cut at that site. The cut is then repaired using the cells’ own machinery, either via non-homologous end joining, which introduces base deletions or insertions, or by homology-directed repair facilitated by a repair template. The latter pathway is far less error-prone due to the homology, or similarity, required between damaged and repair template sequences. These techniques are the driving force behind the expansion of the CRISPR industry, currently at an annual increase of 24% from $550 million in 2018.
For a genome-editing technology, the most obvious place to start is with potential treatment of genetic diseases. The root problem of hereditary disease lies in the intertwining A, T, G and Cs that form the alphabet of our genetic code. The CRISPR/Cas9 system can facilitate the correction of genetic defects by inducing DNA repair at the desired sequence, thus leading to several therapeutic opportunities.
The treatment of monogenic conditions is currently the most widely explored area of gene therapy with CRISPR. One example can be seen in the treatment of Duchenne muscular dystrophy, a severe condition triggered by the mutation of the X-linked dystrophin gene, causing the degeneration of skeletal muscle. Repair via non-homologous end joining produces an unpaired nucleotide at the point of cleavage, allowing the reframing of the mutated gene. Interestingly, the multinucleate structure of skeletal muscle optimises it for CRISPR editing as studies show that a correction of only 15% of nuclei can completely restore levels of dystrophin.
Another example of a monogenic disease, cystic fibrosis is an autosomal recessive disorder that arises from the mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, resulting in the progressive loss of lung function. Following the successful repair of the CFTR gene in intestinal stem cell organoids, CRISPR research is posed to cure the debilitating disease. Homology-directed repair proves to be an unlikely pathway due to the low division rate of airway epithelial cells, therefore one study investigated the use of the ligase inhibitor enzyme Scr7 in mouse embryos to favour the pathway over non-homologous end joining.
But what about multigenic diseases such as cancer? A recent study employed CRISPR/Cas9-mediated editing of fusion oncogenes, formed from the joining of two genes, to repress tumour growth and induce genomic deletion exclusively in cancer cells. Researchers have also begun to investigate the deliberate mutation of Cas9 to eliminate its cutting activity in a form called nuclease dead Cas9 (dCas9). dCas9 can be directed to a specific locus, where it binds to its target gene to suppress transcription, and may be used to down-regulate oncogenes.
It goes without saying that scientific revolution of this calibre cannot arise without its complications. In vivo studies use adeno-associated virus to deliver the CRISPR/Cas9 complex to the target cells, but the Cas9 gene must be small enough to fit within the viral capsid. Studies have found use of a smaller Cas9 variant improves the efficiency of delivery to target cells. Off-target gene modification is being battled by experimentation with an alternative form of Cas9 called Cas9 nickase (Cas9n). Cas9n combines two gRNAs for increased accuracy of target site recognition. This, however, is a double-edged sword ‒ the paired gRNAs may prove to be too big to be packaged into a single vector, and the delivery of the complex is once again compromised.
The CRISPR toolbox continues to diversify, reaching new frontiers in diagnostics and treatment. However, it has occurred to many that this is merely the tip of the iceberg. Studies have found that editing of human embryonic cells can result in the loss of a segment or whole chromosome, an exemplification of how germline editing introduces permanent, yet unpredictable effects into future generations. A mere 6 years after Charpentier and Doudna’s publication, an uproar surfaced in relation to Chinese scientist He Jiankui’s attempt to engineer HIV-resistance in human embryos, which is yet to be confirmed as successful. It will be essential to tread carefully and thoughtfully as we delve deeper into CRISPR-based solutions to disease.
CRISPR. 6 letters.1 revolution. What’s next?