CRISPR-Cas12a mediated combinatorial knock-out of multiple epigenetically silenced genes promotes pro-tumorigenic properties in colon cancer evolution
Embargo until
2027-08-01
Date
2023-06-21
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Publisher
Johns Hopkins University
Abstract
Genome-wide epigenetic alterations are important for the development of cancers. In any given cancer tissue, multiple important developmental regulators undergo epigenetic silencing. Similar epigenetic alterations are observed in normal physiological aging. The cumulative effects of multiple epigenetically silenced genes (ESGs) on tumor development remains an unaddressed aspect of cancer biology, due to the lack of tools for targeting multiple genes, tractable biological models, and assays to measure tumor development. Here, we established a BrafV600E ¬driven ex vivo mouse proximal colon organoid model of colorectal cancer (CRC) to follow tumor progression and identify the effect of multiple ESGs on tumor progression. We used a class V CRISPR system that uses Cas12a to produce CRISPR RNA (crRNAs) targeting multiple ESGs from an array of precursor-crRNA (precrRNA). To address how genes altered by epigenetic changes cumulatively impact cancer initiation, genes undergoing simultaneous epigenetic silencing in human colon cancers were studied. We knocked out combinations of such genes using a newly designed CRISPR-Cas12a system. To use the system, our CRISPR arrays contained semi-random combinations of four guide RNAs. Organoids transduced with our combinatorial lentiviral library were screened for Wnt factor independent growth to assess tumorigenic potential. Combinatorial guide arrays were amplified from the organoids that were able to survive in Wnt factor deficient medium and subsequently sequenced. Overrepresented guide RNAs combinations were analyzed. We demonstrated that in comparison to individual gene knockout, targeted knockout of multiple ESGs in the Wnt and senescence pathways, including the key anti-differentiation gene Cdx2, in the organoids have synergistic effects on inducing Wnt-independent growth and tumor initiation. Inactivating multiple ESGs simultaneously in the absence of oncogene induction sufficiently increases stemness potential. Finally, in preliminary analyses, we identified combinations of guides, mainly driven by Cdx2 and Apc targeting, that provide Wnt independent growth. Our preliminary analyses suggest that functional screening by inactivation of multiple genes by CRISPR approaches can identify the role of simultaneous inactivation of multiple genes in the early stages of tumor development.
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Keywords
DNA methylation, Colorectal Cancer, CRISPR-Cas, Organoids, Organoids