Drug Discovery
skin and hair follicle abnormalities, resulting in hair loss, and depletes specific stem cell popula- tions in the intestine leading to dramatic weight loss26. Importantly, these effects were completely reversible upon doxycycline removal and Brd4 re- expression. Two years following this study, near- identical phenotypes were reported in mice treated with optimised BET inhibitors, CPI203 and I- BET15127.
These results highlight the potential of RNAi mice, which can be used to predict side-effects with- in susceptible tissues and organs and evaluate thera- peutic indices of pharmacological inhibitors a priori.
CRISPR/Cas9: A Game-Changing Tool As you can see, advances in RNAi have enabled us to model drug therapy. Now, by successfully har- nessing CRISPR/Cas9 technology for genome edit- ing, we can induce targeted, disease-specific muta- tions in the same RNAi animals, thus enabling the systematic interrogation of mammalian genome function in specific disease states.
The potential of CRISPR/Cas9 has been widely reported, not just in the science press but the pop- ular press as well and is fast becoming the pre- ferred methodology for engineering mice. From cancer to Huntington’s, scientists are using CRISPR/Cas9 to generate mouse models of disease and some scientists have begun using the CRISPR- Cas9 system to generate other animal models besides mice. To put things in context, it used to take 12-18 months to make a transgenic mouse using traditional techniques. CRISPR/Cas9 does it in anywhere from three to nine months. Using CRISPR to perform genome editing is actually not a new concept, as TALENS and Zinc finger nucleases have been around for decades28,29. However, the CRISPR/Cas9 system is much more flexible and efficient, making it faster and cheaper to use30-32. It consists of a Cas9 enzyme that snips through DNA like a pair of molecular scissors and a small RNA molecule that directs the scissors to a specific sequence of DNA. Following DNA cleavage, there are two different kinds of repair mechanisms that can be used to introduce a desired mutation into a cell’s genome: the homology-directed repair (HDR) pathway which uses a DNA template to copy and repair and the non-homologous end-joining (NHEJ) system. HDR is precise but occurs at very low frequency in mammalian cells. The NHEJ is more efficient but less precise.
By delivering specific DNA templates, scientists can use the HDR pathway to make specific gene modification. They have tried to make this process
Drug Discovery World Fall 2017
more precise and more efficient by using proteins to inhibit the most dominant repair protein of NHEJ and inserting a gene into a predefined posi- tion of the genome in mouse cells33. Another lab has been working on trying to improve the utility of the HDR pathway by using Scr7, which appears to enhance the efficiency and specificity of CRISPR by inhibiting DNA ligase 5. In fact, the group found a 19-fold increase in HDR efficiency with Scr733.
Both of these approaches have been used to develop many different CRISPR-generated disease models, either by NHEJ which generates random mutations to inactivate genes or HDR which can replace portions of a gene, disrupting it with an artificial piece of DNA or even replacing it with alternative gene versions (ie human gene)34-36. While the applied use of Cas9 is now routine in many research labs, and even being used in a few clinical studies, there are other naturally-occur- ring Cas proteins. As these become better charac- terised they could potentially be incorporated into other systems.
The marriage of RNAi and CRISPR/Cas9
The literature often casts the discussion around these two technologies as competing technologies. But my view and the view of others I work with is that they really are complementary. Cancer offers a good example of the benefits of combining RNAi and CRISPR/Cas9 to animal models. To genetically engineer a cancer model, multiple mutations must be engineered in the same animal in order for can- cer to occur de novo. Traditionally this was accom- plished by interbreeding mice with specific muta- tions to one another for years on end until the desired multi-allelic model was obtained, a labour- intensive and expensive process. CRISPR/Cas9 combined with RNAi allows us to accelerate this. For instance, we can take pre-engineered mouse embryonic stem (ES) cells and, using CRISPR/Cas9 in vitro, generate multiple modified alleles that together will give rise to a specific cancer. We can then sequentially target the same CRISPR-modi- fied ES cells with a specific shRNA and then use those ES cells to make mice. These mice will not only get cancer but they will also have an shRNA(s) to test for therapeutic intervention all in the same animal. This process dramatically speeds up the time it takes to create multi-allelic disease models and validate them.
In another instance, animals harbouring a Cas9 transgene are effective for generating rapid somatic mutations37,38. Using this Cas9 mouse, we can
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