It is rare that the development of a new scientific technique causes just as much furore amongst the general public as it does amongst researchers. The announcement of CRISPR-Cas9 as a new means of achieving high-precision genome editing was one such occasion: in 2013, when it was first announced that the technology had been used to edit mammalian cells, questions of safety, ethics and “designer babies” dominated headlines and incited public debate.
Yet such issues are still a few years off: the CRISPR system is still being honed, with only a few clinical trials announced (LePage, 2017) and even fewer currently underway (Cyranoski, 2016a). It is even less likely that any trials involving permanent, germline edits will be approved in the next few years. Rather, in the short term, CRISPR-Cas9 will have its greatest impacts on basic research.
Ethics, safety, and practicality preclude researchers from investigating disease in human subjects. Thus, disease models are used to gain an understanding of the underlying mechanisms that cause pathology. Early-stage models usually involve in vitro cell or tissue cultures grown from samples obtained from an affected individual, though there are several limitations: by having just a subset of the complex network that is the human body, the effects of a drug may not be properly quantified by just testing it on one specific tissue. Similarly, some diseases and syndromes are pleiotropic, affecting many different tissues or organs. Here, in vitro studies may facilitate study of a component of a disease, but not the condition in its entirety.
Though controversial, animal studies have proved more useful in many respects than in vitro cultures. However, they too have limitations – some conditions that are relatively common in humans, such as multiple sclerosis, lack exact equivalents in animals and though model systems exist to study the diseases – experimental autoimmune encephalitis in the case of MS – important differences exist that limit transferability of results.
That’s where genome editing plays a role. By introducing specific changes to DNA, be they in cultured tissue or a live animal, experimenters have greater power to mimic a disease as they would be in patients.
CRISPR’s power to enhance in vitro disease modelling is particularly prominent when paired with another recent advancement in biology: induced pluripotent stem cells (iPSCs). These cells are taken from a patient and then transfected with four key pluripotency genes (the so-called Yamanaka factors) such that they can then differentiate into any cell in the human body. iPSCs are particularly useful when trying to generate organs with many different tissue types, overcoming one of the primary limitations of in vitro cultures.
Coupled with CRISPR-Cas9, researchers now have the unprecedented ability to take diseased cells and edit them with high efficiency, both to understand the disease and to develop new therapeutics. This dual approach was used when investigating polycystic kidney disease (PKD) (Freedman et al., 2015). Here, researchers used human-induced-pluripotent-stem-cell derived kidney cells (hPSC-KC) to generate “organoids”, cultures that mimic organs more closely than normal cultures but lack the full complexity of organs. They then used CRISPR-Cas9 to generate knockouts of PKD1 and PKD2, both of which have been implicated with the condition. The researchers found that these loss-of-function mutations lead to the formation of cysts, meaning the system can now be used in further studies of kidney diseases.
Importantly, the high efficiency of the CRIRSPR-Cas9 system means that animal models are also easier to generate as problems such as chimerism are more readily overcome. Thus far, over 1,000 mutant strains of mice have been engineered (Simmons, 2008) though with the advent of CRISPR technology this is likely to dramatically increase.
Motor neuron disease (MND; also termed amyotrophic lateral sclerosis, or ALS) is a neuropathology that results in the gradual weakening of muscles. Despite intensive research, its cause is still unknown – over 90% of cases are idiopathic, with the remaining sufferers having some inherited form of the condition. Even so, using next generation sequencing (NGS) over twenty genes associated with the condition have been identified and it is now understood that many alleles of these genes interact to create a patient-specific phenotype (Liu, 2017). CRISPR can then be used to create mice strains with particular combinations of these alleles, facilitating more accurate modelling and even bringing us a step closer towards “personalised medicine”.
Monogenetic diseases are relatively rare. Rather, disease phenotypes are usually the product of multi-gene interactions, with different combinations of alleles conferring different degrees of disease severity or likelihood of getting the disease in the first place. Such intricacy is hard to study in animal models, and even when older genome-editing technologies such as Zinc Finger Nucleases (ZFN) or Transcription Activator-Like Endonucleases (TALENs) were used, researchers were still limited to editing one gene at a time. However, recent studies show that CRISPR can be used to in a multiplex and edit several genes at once. Already, this has been used to model pancreatic cancer in mice (Maresch et al., 2016). The study showed that mutational load was of high importance in determining the probability of developing cancer, but importantly which combinations were most dangerous.
Preventative medicine will also benefit from the fine-tuning facilitated by CRISPR. Ferrets are commonly used to study the transmission and replication of viruses that cause respiratory infections, namely influenza viruses (Belser et al., 2011). Though initially due to their tractability and similar physiology to humans, the publication of both the ferret transcriptome and genome has made them even better model organisms as they are now primed for use in transgenesis (Enkirch and von Messling, 2015). CRISPR-Cas9 can then be used to alter the ferret’s physiology, making it even more similar to that of humans and potentially improve vaccine development.
Disease models of every kind – in vitro or animal, mouse or monkey – have been instrumental in the development of new therapeutics and cures. Nevertheless, CRISPR allows those developments to be brought even further by allowing the creation of new models for diseases that previously lacked any. CRISPR-Cas9 is still in its infancy, but as the technology progresses its potential use in modelling complex diseases becomes even greater.
Belser, J.A., Katz, J.M. and Tumpey, T.M. (2011). The ferret as a model organism to study influenza A virus infection. Disease Models and Mechanisms 4:575-579
Freedman, B.S., Brooks, C.R., Lam, A.Q., Fu, H., Morizane, R., Agrawal, V., Saad, A.F., Li, M.K., Hughes, M.R., Vander Werff, R., Peters, D.T., Lu, J., Baccei, A., Siedlecki, A.M., Valerius, M.T., Musunuru, K., McNagny, K.M., Steinman, T.I., Zhou, J., Lerou, P.H. and Bonventre, J.V. (2015). Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids. Nature Communications 6:8715
Enkirch, T. and von Messling, V. (2015). Ferret models of viral pathogenesis. Virology 479:259-270
LePage, M. (20th May 2017). Boom in human gene editing as 20 CRISPR trials gear up.
Liu, E.T., Bolcun-Filas, E., Grass, D.S., Lutz, C., Murray, S., Shultz, L. and Rosenthal, N. (2017). Of mice and CRISPR. EMBO Reports 18:187-193
Maresch, R., Mueller, S., Veltkamp, C., Ollinger, R., Friedrich, M., Heid, I., Steiger, K., Weber, J., Engleitner, T., Barenboim, M., Klein, S., Louzanda, S., Banerjee, R., Strong, A., Stauber, T., Gross, N., Heumann, U., Lange, S., Ringelhan, M., Varela, I., Unger, K., Yang, F., Schmid, R.M., Vassiliou, G.S., Braren, R., Schneider, G., Heikenwalder, M., Bradley, A., Saur, D and Rad, R. (2016). Multiplexed pancreatic genome engineering and cancer induction by transfection-based CRISPR/Cas9 delivery in mice. Nature Communications 26: doi: 10.1038/ncomms10770
Simmons, D. (2008). The use of animal models in studying genetic disease: Transgenesis and induced mutation Nature Education 1:70