Uncovering cell-intrinsic restrictions to CRISPR-Cas9 gene editing

Project Summary Multipotent, tissue-specific stem cells (“adult stem cells”) are major targets for therapeutic gene editing because of their longevity and capacity to differentiate into specialized cell types. However, the site-directed genetic modification of adult stem cells is inefficient in vivo. Further, no robust characterization of genome

editing efficiency across a complete cellular lineage has been performed to understand cell-intrinsic restrictions to gene editing in undifferentiated and differentiated cell types. Much of my postdoctoral work has focused on developing virus-like particles as a delivery vehicle for pre-assembled CRISPR Cas9-sgRNA ribonucleoprotein

(RNP) complexes for editing of primary human cells ex vivo. The primary aims of this proposal are therefore: 1) to characterize the the baseline relative gene editing and base editing efficiencies of cell types derived from a complete cell lineage (hematopoietic cells) containing multipotent and differentiated cell types, 2) leverage

CRISPR-Cas screens in adult stem cells (hematopoietic stem cells) and terminally differentiated cells (T cells) to identify genetic factors that modulate gene editing efficiency, 3) couple CRISPR-i and Perturb-seq to uncover genetic factors responsible for maintaining adult stem cell homeostasis following gene editing and 4)

utilize virus-like particles packaging Cas9 RNP complexes to achieve genome editing of adult stem cells in vivo. The proposed research will provide considerable insight into the basic biology underpinning gene editing determinants in undifferentiated and differentiated primary cells, and the feasibility of using Cas9 RNPs to

mediate therapeutic gene editing in multipotent cells in vivo, using hematopoietic stem cells as a model cell type. Significant findings relevant to the fields of stem cell biology, DNA repair biology, therapeutic genome editing are expected. Areas of additional scientific training that will enable successful completion of this

proposal are knowledge of primary cell culture, experience conducting genome-wide CRISPR-Cas screens and single-cell RNAseq analysis. The mentored phase of the award will be supervised by Dr. Jennifer Doudna, a world-leader in genome editing technology. Dr. Doudna, and all other collaborators on this project are located

at UC Berkeley or in the greater San Francisco Bay Area scientific community. During the mentored phase of this project I will continue scientific professional development activities to improve as a scientific leader and gain a thorough grounding in topics essential for running my own independent research group. I will also

continue presenting my research at national and international conferences (likely remotely while COVID-19 precautions are in effect). When combined with the excellent research environment at UC Berkeley and the Innovative Genomics Institute, I have an outstanding opportunity to complete my foundational training as I

begin my transition to independence.