Collaborative science was something Parrish aspired to because it “literally makes my science better,” she exclaims. However, “so many collaborations are transactional. I wanted to take that aspect out and make it really collaborative and fun,” notes Parrish. To this end, she worked with Dr. James Thomas, a postdoc at the Bradley Lab, part of the Departments of Basic Research and Public Health, to develop a CRISPR-Cas9 method to directly study paralog compensation at the genomic level. Aiming to specifically identify synthetic lethal paralogs that could serve as potential targets for lung cancer drugs, the research team focused on duplicated genes – paralog families consisting of just two genes. Parish et al. Then over 1,000 human paralog families (>2,000 genes) were knocked out, targeting each gene paralog individually or in combination with its paralog pair, using paired guide RNAs for a paralog gene interaction mapping library ( pgPEN) consisting of over 33,000 paired guide RNAs (pgRNAs) ). Researchers tested their pgPEN library in lung adenocarcinoma cells and identified paralogues essential for cell growth and viability using next-generation sequencing to assess the abundance of pgRNAs after a 21-day competitive growth screen. Here, small amounts of pgRNAs would indicate genes that promote the growth of cancer cells. Among these top hits were important cell cycle regulators and MEK1/MEK2, confirming that their previously observed discrepancy between genetic and drug data was indeed due to paralog redundancy. Researchers then experimentally validated the key synthetic lethal paralog pairs by developing a fluorescent reporter-based competitive fitness assay in lung cancer cells, verifying that cells with both paralog genes knocked out were less competitive than those with one paralog -Gen was missing.
To understand which synthetic lethal paralogs are shared between cell types and which are cell lineage specific, Parrish et al. repeated this pgPEN screen in HeLa cervical cancer cells. The researchers found that many of the best synthetic lethal pairs were shared between cervical and lung cancer cells, but others were cell-type specific. Interestingly, the cell lineage-specific synthetic lethal interactions could not be explained by differences in expression between the two lineages, showing that paralog dependencies are modified by the cellular context. In addition, they identified several gene-paralog pairs that, when knocked out, promoted cell growth, indicating that these paralog families likely had tumor-suppressive functions that required loss of the family to reveal the cellular phenotype. Interestingly, they identified four tumor suppressor paralogues in lung cancer cells, none of which were shared with the six tumor suppressor paralogue pairs identified in cervical cancer cells. Overall, this work identified putative paralog gene targets for lung cancer therapies in addition to revealing tumor suppressor paralogs. More generally, this study highlights the importance of duplicate paralog genes and encourages scientists to consider how ubiquitous these functionally redundant genes might be in their own research, and provides researchers with a method to test this question in their system.
In addition to deciding to publish this work in an open access journal, Parrish is working to further improve the accessibility of her research by now working to publish the computational methods and code generated for this project’s data analysis . Aside from the science, Parrish describes that one of the amazing aspects of working on this project was conducting this research in the currently all-female Berger Lab, which fosters an inclusive, diverse environment that Parrish felt was critical to advancing her own science. This research experience further emphasized the “importance of funding the basic research that really drives discovery,” but also “funding and supporting diverse people in the science community,” notes Parrish. Beyond the Berger Lab, Parrish has worked to promote diversity and inclusion by establishing a diversity program within UW Genome Sciences, which she sees as a source of motivation during the ups and downs of her own research.
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