Replication Study of Off-Target Assays for Guide RNAs in CRISPR-Based Therapeutics - PROJECT SUMMARY/ABSTRACT. This application is being submitted in response to the Notice of Special Interest (NOSI) identified as NOT-RM- 24-009. In vivo gene editing still faces substantial challenges, especially when it comes to safety, efficacy, and delivery. In particular, the variability in off-target effects of CRISPR-based therapeutics poses a significant challenge for their clinical application. By systematically comparing inter-lab studies, this proposal will provide critical insights into the reproducibility and reliability of off-target assays. The findings could inform best practices for assay and reagent selection, ultimately enhancing the safety and efficacy of CRISPR-based therapies. Using contract research organizations (CROs), this study introduces a novel approach to comparing off-target assays with a clinically relevant non-viral formulation. This innovative comparison will provide a deeper understanding of the factors influencing assay outcomes, paving the way for more reliable and standardized methodologies in preclinical research. In the first aim, we will compare off-target nomination assays using W53* guide RNAs for our lead Trailblazer formulation in our U19 SCGE parent award. The CRO will purchase or acquire reagents from the labs within the parent award and perform genome-wide off-target nomination assays at their facility. The results from the CRO will be compared against completed CasOFFinder and CHANGE-Seq-BE nomination assays already completed. We will compare the sensitivity, specificity, and reproducibility of the assays. In the second aim, we will compare short-read sequencing around nominated off- target sites in W53* patient-derived retinal cells. The CRO will deliver the editor to a human biological model of the target cell type, retinal pigment epithelial cell differentiated from induced pluripotent stem cells (iPSC-RPE). The CRO purchase or acquire reagents from the labs within the parent award and perform sequencing at their facility. Again, we will compare the sensitivity, specificity, and reproducibility of the assays. This replication study will provide critical data on the variability of off-target assays, informing best practices for CRISPR-based research. The results will enhance the reliability and reproducibility of off-target assays, supporting the development of safe and effective CRISPR-based therapeutics.
