UC Berkeley

CRISPR genome editing tools continue to propel major advances in biological research and offer new therapeutic avenues to treat genetic diseases. For in vivo therapeutic or research applications, there is the additional hurdle of safe and effective delivery of CRISPR effectors intracellularly. For gene therapies, delivering CRISPR ribonucleoproteins (RNPs), rather than DNA or RNA, can result in comparably efficient genome modification while mitigating undesirable clinical outcomes, such as off-target editing and increased immunogenicity, that can arise from extended gene editor expression. First, we discuss progress towards engineering next-generation CRISPR RNPs for mammalian genome engineering with broader and efficient genomic targeting capabilities, improved editing specificity, packability and nuclease activity, and outline key considerations and remaining critical challenges. Next, we compared two delivery strategies, electroporation and enveloped delivery vehicles (EDVs), to investigate the Cas9 dosage requirements for genome editing. Using fluorescence correlation spectroscopy (FCS), we determined that >1300 Cas9 RNPs per nucleus are typically required for productive genome editing. EDV-mediated editing was >30-fold more efficient than electroporation, and editing occurs at least two-fold faster for EDV delivery at comparable total Cas9 RNP doses. We hypothesize that differences in efficacy between these methods result in part from the increased duration of RNP nuclear residence resulting from EDV delivery. Our results directly compare RNP delivery strategies, showing that packaged delivery could dramatically reduce the amount of CRISPR-Cas9 RNPs required for experimental or clinical genome editing. Lastly, to identify prominent Cas9 RNP delivery bottlenecks and commonalities, we utilized spatiotemporally resolved proteomics to compare RNP delivery by electroporation, EDVs, and in trans cell penetrating peptides (PERC), in HEK293T and T-cells. This workflow uncovered cell-type differences in uptake for EDVs and PERC. Regardless of delivery strategy, Cas9 associates with endogenous RNA binding proteins and localizes to the nucleoli prior to degradation. This method enables flexible comparison of Cas9 RNP delivery strategies, and the impact of cell-type on uptake mechanism. We conclude with an outlook on the future of CRISPR RNP delivery and contextualize key remaining challenges. Together, the projects presented in this dissertation contribute to the growing understanding of CRISPR RNP engineering and delivery strategies.