Browsing by Author "Ostrin, Alex"

The bacterial CRISPR-cas9 system has revolutionized our ability to manipulate the genome through precise RNA-guided editing. Despite its success in cutting precisely at target loci, RNA-guides also bind to off-target genomic regions, inducing undesired, and potentially genotoxic, double stranded breaks (DSBs). Currently, there exists no method for predicting guide RNA (gRNA) on-target and off-target efficiency with sufficient accuracy for all types of gRNA (i.e. gRNA targeting coding and non-coding regions) across cell-types. In this thesis, I present the development of a high-throughput, pooled GUIDE-seq assay to experimentally profile Cas9 on and off-target cleavage across a diverse set of gRNAs. This assay builds on the established GUIDE-seq protocol, which labels DSBs in vivo with a double-stranded oligodeoxynucleotide (dsODN) tag, enabling genome-wide identification of cleavage sites via amplicon sequencing. As a proof-of-principle, I demonstrated successful GFP knockout (KO) in a reporter cell line using both plasmid transfection and ribonucleoprotein (RNP) nucleofection, validated by flow cytometry and next-generation sequencing (NGS). I then cloned a pilot guide pool and generated a stable cell line expressing the pool via lentiviral infection. A preliminary GUIDE-seq experiment confirmed successful dsODN integration at a target site, though integration rates remain below the threshold necessary for robust off-target detection. This work lays the foundation for a pooled GUIDE-seq pipeline, with future efforts focused on optimizing the assay to generate comprehensive off-target profiles. These data will support the development of a predictive model – GuideScan3 – for the rational design of highly specific gRNAs, facilitating the broader use of CRISPR-cas9 editing in therapeutics and basic science without inducing harmful off-target effects.