Cutting edge Seminar

※  Zoom online/ Please check the URL on “HIGO Cutting-Edge Seminar” at Moodle

 

Speaker: Kosuke Yusa (Professor, Institute for Frontier Life and Medical Sciences, Kyoto University Department of Biosystems Science Lab. of Stem Cell Genetics)

Title: Development and application of CRISPR-KO screening

 

Date&Time: 29  Jul.  (Wed.) 2020, 12:00- 13:00

Venue:  Zoom online/ Please check the URL on “HIGO Cutting-Edge Seminar” at Moodle

 

Abstract:

The advent of the CRISPR system has transformed the landscape of genetic studies. Its high efficiency, simplicity, flexibility and scalability have enabled us to develop a number of different types of genome engineering tools that can be applied in a wide range of organisms. My laboratory has been focusing on developing tools for large-scale functional genomics to comprehensively identify genes and pathways involved in a phenotype of interest – so called, forward genetics. We applied the CRISPR-Cas9 system for the development of genome-wide loss-of-function screening (CRISPR-knockout screening) and demonstrated its high efficiency (Nature Biotechnology, 2014). We then further refined and optimized the technology for more efficient dropout screens (Cell Reports, 2016).

We have applied CRISPR-KO screening in two research areas: oncology and pluripotency. For oncology projects, we aimed to systematically characterize genetic dependencies in cancer cells. As a proof of concept, we first performed a small scale screen in a panel of 5 acute myeloid leukaemia (AML) cell lines and identified ~200 druggable vulnerabilities (Cell Reports, 2016). We have thus far validated 3 promising novel candidates, whose genetic and pharmacological inactivation of these genes induced myeloid differentiation and apoptosis (Nat Commun, 2018). Following the success in the AML screen, we extended this approach to a much larger panel of 324 cancer cell lines (Nature, 2019). We identified approximately 7,500 genes, which are essential in at least one cell line. Integration of multiple datasets revealed ~600 high-priority targets. Of these, Werner RecQ DNA helicase is specifically required for cancers with microsatellite instability. Our analysis provides a resource of cancer dependencies, generates a framework to prioritize cancer drug targets and suggest new targets.

For pluripotency analysis in mouse ES cells, we used Rex1-GFP as a naïve pluripotency marker and performed cell sorter-based screens to identify genes required for general maintenance as well as exit from pluripotency (Cell Reports, 2018). The majority of the previously characterized factors were successfully identified in both screens. We identified that two negative regulators of mTORC1, namely Tsc1/2 and Gator1, modulate exit from pluripotency in a opposite direction, which was mediated by Gsk3. We also performed a similar screen in human iPS cells using Tra1-60 as a pluripotency marker. Known factors such as POU5F1, FGFR1 and PRDM14, as well as novel factors were identified. We are now conducting detailed molecular analysis of these new pluripotency factors.

As exemplified above, CRISPR-KO screening is a powerful unbiased approach to genetically dissect a phenotype of interest. This approach can be applied in various biological processes and would reveal novel mechanisms.