The subject of this presentation brings together two Nobel Prize winning technologies. 2012’s prize for physiology or medicine was awarded for work in developing induced pluripotent stem cells (iPSCs), and in 2020, the prize for chemistry was conferred for the gene modifying technology CRISPR/Cas9. The combination of these tools results in immense opportunities from improved models to identifying novel targets.
iPSCs were developed to fix the limitations with the older tool of embryonic stem cells. By taking differentiated somatic cells and reprogramming them with transcription factors, these cells will revert to a state similar to embryonic stem cells. They can be frozen, thawed, and grown indefinitely, making them highly useful for biological research. For example, iPSCs are used in human tissue modelling in 3D cell cultures and organoids.
CRISPR/Cas9 highjacks the cells’ repair system to force the insertion or deletion of specific genes in a DNA sequence. The combination the CRISPR/Cas9 system with the use iPSCs opens up a world of possibilities for research. For example, Sharma mentioned disease models, therapeutic modalities, high-throughput screening, immune system research, drug discovery, target identification, drug efficacy models, and regenerative medicine, to name a few...
There are however practical hurdles that researchers must jump in order to bring together these tools. First, scientists must work out whether to use plasmids to deliver their guide RNA or ribonucleoprotein (RNP) delivery. Second, what is the most efficient method to deliver it into the iPSCs? The third and fourth hurdles are the time and labour required to use the two systems together. And finally, knock-in efficiency is often low.
Sharma outlined his team’s approach to using the two tools. Firstly, they use RNP delivery due to their better experimental results. For delivery, the team use electroporation as it is most convenient for their lab. To reduce the time and labour costs required, their solution is to improve the efficiency of their DNA amplification enzyme. Furthermore, strategies to enhance homologous directed repair (HDR) in iPSCs, including chemical inhibitors and cold shock treatments, are discussed.
