PROJECT SUMMARY / ABSTRACT The goal of this application is to train Dr. Brian Shy, a physician scientist at the University of California San Francisco, with the skills necessary to become an independently-funded investigator studying, developing, and manufacturing engineered cellular therapies. This research proposal will evaluate CRISPR-based non-viral methods for targeted genome, epigenome, and transcriptome engineering in primary human T cells. This will be applied to understand DNA repair pathway choice and DNA toxicity responses in order to improve site-specific “knock-in” strategies with large therapeutic constructs such as chimeric antigen receptors (CARs), develop safe and efficient methods for multiplexed T cell modifications, and establish Good Manufacturing Practice (GMP) compatible processes for clinical implementation of this diverse toolset. This research is accompanied by a career development and training plan that will build on Dr. Shy's clinical training in Transfusion Medicine and Cellular Therapy, and his prior expertise in the pre-clinical development and manufacturing of experimental cellular therapies. Training will focus on 1) implementation of novel cellular engineering tools, 2) next generation sequencing (NGS) technologies and analysis of large datasets, 3) GMP manufacturing and pre-clinical development, 4) clinical trial design and standard-of-care cellular therapies, and 5) laboratory management, grant writing, presentation, and research strategy skills. This career development and training plan will include a combination of formal coursework, mentored practical training; conference, meeting, and workshop attendance; and guidance from an exceptional team of co-mentors, advisors, and collaborators. Altogether, this award will help prepare Dr. Shy for an independent research career focused on developing improved cellular therapies for treatment of cancer and other human diseases.