Engineering Improved Bipartite Repressors for CRISPR-Based Gene Regulation in Cancer Cells
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Allen, Christopher
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Abstract
CRISPR interference (CRISPRi) has emerged as a powerful technique for exploring gene-phenotype relationships in human cells. The CRISPRi platform works by delivering a bacterial protein, dCas9, to the nucleus of a given cell to decrease the expression of target genes. Although CRISPRi has immense potential, one limitation is that it exhibits high performance variability depending on the repressor domain fused with dCas9. Currently, the best CRISPRi systems utilize single repressor domains from the Krüppel-associated box (KRAB) family, and previous studies found that fusing KOX1-KRAB with other repressors improved CRISPRi performance. Since then, others have identified hundreds of novel repressor domains, but no study has examined whether these novel repressors have improved activity when fused together or combined with KRAB domains. To test potential fusions, we first quantified the repressive capability of twenty novel individual human protein domains using a fluorescence-based reporter assay in HEK293T cells, in which we discovered that nearly all exhibit compatibility with CRISPRi. We then tested combinations of these new repressors with KRAB domains and identified several that outperform the current best-in-class repressors for some endogenous gene targets. These improved CRISPRi repressors would be impactful for numerous applications, such as discovery of genes essential for cancer cell proliferation.
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Undergraduate Thesis