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JAMA
SCIENTIFIC DISCOVERY AND THE FUTURE OF MEDICINE
Biology and clinical medicine are in the midst of a remarkable revolution. Technical advances in DNA sequencing have given scientists access to the molecular code governing each individual’s unique characteristics, including disease susceptibility and drug sensitivity. This remarkable knowledge could only inform researchers and clinicians because tools to act on the data by making targeted changes to the human genome were too expensive and cumbersome for widespread practical use. Nonetheless, the compelling promise of truly personalized medicine spurred the quest for methods to achieve precision genomic manipulation.
In an interesting twist of scientific fate, basic research on an adaptive immune system in bacteria led to the breakthrough genomic engineering technology known as CRISPR-Cas9. Experiments focused on understanding how bacteria acquire resistance to viruses, using genomic loci known as clustered regularly interspaced short palindromic repeats (CRISPR), uncovered the activity of the CRISPR-associated (Cas) RNA-guided DNA-cleaving enzyme called Cas9. Cas9 generates double-stranded DNA breaks at sites matching the sequence of a guide RNA molecule that was designed in the laboratory to include features necessary for both Cas9 binding and DNA recognition (Figure).1 When introduced into animal cell nuclei, the Cas9 protein and RNA-guided complexes trigger site-specific genome editing by generating double-stranded DNA breaks that are repaired by the cell’s natural machinery.
Figure. Genomic Engineering Mediated by CRISPR-Cas9 Technology The natural dual-tracrRNA:crRNA complex was engineered as a single-guide RNA (sgRNA) that retains 2 critical features: the 20-nucleotide guide sequence that specifies DNA binding by Watson-Crick base pairing and the hairpin structure that recruits Cas9. Changes to the 20-nucleotide guide sequence serve to program CRISPR-Cas9 to target a DNA sequence adjacent to an NGG sequence motif (the protospacer-adjacent motif, PAM). Cas9-induced double-stranded DNA breaks are repaired either by nonhomologous end joining (NHEJ) or homology-directed repair (HDR). The NHEJ typically generates a small insertion or deletion at the site of the original break, which can be used for gene disruption. HDR can introduce new genetic information at the site of the break.
Jennifer A. Doudna, PhD1,2,3
This discovery has triggered a veritable revolution as laboratories worldwide have begun to introduce or correct mutations in cells and organisms with a level of ease and efficiency not previously possible. In just 2½ years, more than 500 articles have reported a vast array of applications of the CRISPR-Cas9 system. Recent reviews describe the advent of this technology and its widespread use.2,3 This Viewpoint focuses on advances that underscore the potential of the CRISPR-Cas9 system to directly affect the study, treatment, and prevention of human disease.
STUDYING DISEASE: ENGINEERING PRECISE MODELS
The lack of efficient, inexpensive, fast-to-design, and easy-to-use precision genetic tools has long been a limiting factor for the analysis of gene functions in animal models of human disease. Efficient genomic engineering to enable targeted genetic changes both in somatic cells and in the germlines of a wide variety of animals would facilitate pharmacological studies and the understanding of human disease pathways in ways not previously attainable. One of the most substantial types of genomic abnormality occurs due to rearrangement of nonhomologous chromosome segments. In the past, modeling such chromosomal translocations in adult animals has been challenging due to the requirement for complex manipulation of DNA in germline cells. The CRISPR-Cas9 system enables induction of exact chromosomal translocations in somatic cells, thereby producing a much more robust and representative animal model of carcinogenesis.
A recent exciting use of this technology generated disease-associated chromosomal translocations in adult animals.4 This study focused on the fusion between genes encoding echinoderm microtubule-associated like protein 4 (EML4) and anaplastic lymphoma kinase (ALK), which occurs when there is an inversion of the short arm of chromosome 2. The resulting EML4-ALK fusion oncogene is a signature of some human non–small-cell lung cancers and makes these cells sensitive to ALK inhibitors. Thus, the ability to generate this specific translocation creates an opportunity to study the development of tumors in response to such genetic rearrangements. By using virus-mediated delivery of the CRISPR-Cas9 system to induce the Eml4-Alk fusion in adult mice, animals developed lung tumors harboring the Eml4-Alk inversion.4 The tumors produced the Eml4-Alk fusion protein, displayed histological changes and molecular properties typical of ALK-positive human non–small-cell lung cancers, and responded to treatment with ALK inhibitors. Notably, the CRISPR-Cas9 system can also be used to introduce inactivating point mutations in cancer-associated genes in adult tissues. For example, it was possible to induce mutations in Pten and p53, which are defective in a variety of human cancers, in the livers of mature mice, leading to liver tumors containing genetic lesions in the targeted genes.5 These findings demonstrate the power of the CRISPR-Cas9 technology to model human cancers in mice and other organisms.
TREATING DISEASE: CORRECTING POINT MUTATIONS
The CRISPR-Cas9 system provides the precision required to correct point mutations in patient-derived tissues. A promising example of this was performed using organoids, which are multicellular bits of tissue, grown in the laboratory from animal or patient stem cells. By expanding individual mouse or human intestinal stem cells in culture, stable epithelial organoids can be generated that exhibit properties of the tissue of origin. As a case in point, epithelial organoids derived from normal individuals swell in response to increased cyclic adenosine monophosphate (AMP) levels due to the opening of the cystic fibrosis transmembrane conductor receptor (CFTR). Notably, this response is lost in organoids derived from patients with cystic fibrosis in which the CFTR gene contains an inactivating point mutation. By using the CRISPR-Cas9 system to correct the CFTR locus in cystic fibrosis–derived stem cells by homology-directed repair, the functional form of CFTR can be expressed. In clonally expanded organoids generated from these edited cells, the swelling response to cyclic AMP was observed, providing proof of concept for gene correction in patient-derived primary adult stem cells.6 This approach illustrates the potential of the CRISPR-Cas9 technology to treat patients who have diseases caused by single-gene hereditary defects.
Recent experiments in adult mice underscore the possibilities for future use of this technology in patients. For example, mutation of the gene encoding fumarylacetoacetate hydrolase (FAH), the last enzyme in the tyrosine catabolic pathway, gives rise to the fatal genetic disease known as hereditary tyrosinemia type I (HTI). In a mouse model of HTI, the CRISPR-Cas9 system was used to induce correction of the disease-associated FAH mutation in the liver. Delivery of components of the CRISPR-Cas9 system by high-pressure injection of plasmid DNA encoding the Cas9 enzyme and guide RNA resulted in expression of the wild-type FAH protein in 1/250 liver cells.7 Expansion of FAH-positive hepatocytes rescued the body weight loss phenotype in these animals. Such results demonstrate that CRISPR-Cas9-mediated genome editing is possible in adult tissues and has the potential to treat and potentially even cure human genetic diseases.
PREVENTING DISEASE: MOVING BEYOND GENETIC DISORDERS
A key property of Cas9 is its ability to bind to genomic sites defined by the guide RNA sequence and the short DNA sequence protospacer adjacent motif, allowing applications beyond permanent modification of DNA. In particular, a catalytically deactivated version of Cas9 (dCas9) has been repurposed for efficient gene regulation in mammalian cells. Recent studies in which the Cas9 system was used to turn on expression of individual or sets of genes demonstrate the potential use of this form of the CRISPR-Cas9 system to control gene expression in human cells, which could be used to replace or augment diseased tissues. Engineered dCas9 activation complexes can be used to trigger expression of multiple genes simultaneously and to upregulate long intergenic noncoding RNA transcripts that could affect cell and tissue characteristics.8,9 CRISPR-associated RNA scaffolds provide a powerful way to construct synthetic gene expression programs for a wide range of applications, including rewiring cell fates or engineering metabolic pathways.
CONCLUSIONS
The advent of CRISPR-Cas9 technology underscores the importance of basic research for advancing medicine. Once the molecular mechanism of CRISPR-Cas9 was understood, it could be harnessed for applications not previously imagined. Ongoing research focuses on determining Cas9-mediated gene editing specificity, as well as increasing the frequency of homology-directed DNA cleavage repair. Furthermore, methods for delivering Cas9 and its guide RNAs into cells need to be tested in disease-relevant tissues and animal models. The era of genome editing raises ethical questions that will need to be addressed by scientists and society at large. How should such a powerful tool be used to ensure maximum benefit while minimizing risks? It will be imperative that nonscientists understand the basics of this technology to facilitate rational public discourse. Regulatory agencies will also need to consider how best to foster responsible use of CRISPR-Cas9 technology without inhibiting appropriate research and development.