Shannon Stein

•Design and execute high-throughput screens in hepatoma cell lines to identify lead gRNAs for new targets. • Support high-throughput screening efforts for partnership programs • Developed and optimized a high-throughput screening workflow in liver cell lines to enable 3-week durability studies, identifying gRNAs and CRISPR-based epigenetic editors that effectively and durably knockdown target mRNA. •Development, optimization, and validation of assays such as RT-qPCR, bisulfite amplicon seq, and NGS library prep for multiple target genes in human and murine cell lines, primary cells, and nonhuman primate in vivo samples.

• Perform hit validation experiments in primary human, cynomolgus, and mouse hepatocytes, assessing lead gRNA potency and CRISPR-based epigenetic editor durability via RT-qPCR, and evaluating on-target DNA methylation profiles using bisulfite amplicon sequencing. •Develop and execute high-throughput CRISPR-based screens to assess the specificity and activity of many different Cas protein variants and gRNAs across cell types for external and internal programs. •Conduct lead identification optimization and validation studies to enable in vivo studies. •Assay development and optimization

• Determined the optimal ASO knockdown window of lncRNAs in a lung cancer cell line. • Strategically designed gapmer-based ASOs that specifically target the flexible and accessible regions of lncRNAs. • Successfully synthesized near-infrared fluorescent dye-labeled northern probes specific to the lncRNA MALAT1 through an in vitro transcription process. •Applied copper-free click chemistry to effectively attach the dye labels onto the RNA probes, enabling their fluorescent detection in succeeding experiments.

I completed my master's thesis under the mentorship of Dr. Chase Weidmann, where I studied long non-coding RNAs (lncRNAs) that affect the NRF2/KEAP1 pathway in lung cancer. • I adapted a reporter assay to monitor NRF2 activation in a reporter cell line. • Identified novel lncRNAs that exhibited co-activation with NRF2 in lung adenocarcinoma cell lines with qRT-PCR. • Utilized in-cell chemical probing, specifically SHAPE-MaP, to accurately predict RNA secondary structure and identify flexible and available regions of the lncRNAs to target with small molecules in succeeding experiments.

I completed my undergraduate thesis under the mentorship of Dr. Douglas Swanson and Dr. Ajit Sharma, where I synthesized and characterized a mixed-surface generation 4 polyamidoamine (PAMAM) dendrimer. • Synthesized and purified a variety of mixed-surface PAMAM dendrimers. • Achieved high levels of purity with size exclusion chromatography and dialysis. • For characterization, I preformed acidic native PAGE, SDS-PAGE, IR spectroscopy, isoelectric focusing, 13C NMR, and RP-HPLC .

Thesis: Long noncoding RNAs that affect the NRF2/KEAP1 pathway in lung cancer

Thesis: Synthesis and characterization of a mixed surface generation 4 polyamidoamine dendrimer