UC San Diego

Understanding and manipulating biological systems at a molecular level is crucial for therapeutic drug development. Perturbations introduced through various methods allow researchers to observe effects on cellular processes, providing insights for therapeutic intervention. Old school methods like chemical treatments, transfection, and genetic modifications often face limitations in efficiency, precision, and cost. Advances such as RNA interference (RNAi) and CRISPR/Cas9 have revolutionized gene editing, with CRISPR/Cas9 enabling precise and permanent genetic modifications. These advances allowed for a pooled approach where sgRNAs can target multiple genes within a single sample. We propose to recreate an innovative optical pooled screen method with in-situ sequencing (ISS) to directly sequence guide RNAs (sgRNAs) using the CROPseq vector, eliminating barcode assignment errors and enhancing imaging throughput. Two major experiments were conducted: identification of rolling circle products to confirm amplification of sgRNA and achieving sequencing by synthesis. Both experiments have multiple iterations that target both an endogenous gene and a transduced sequence. This method surpasses previous techniques by efficiently linking genotype to a wide range of phenotypes, providing a powerful tool for high-resolution, high-throughput genetic screening.