# Uncovering cell-intrinsic restrictions to CRISPR-Cas9 gene editing

> **NIH NIH K99** · UNIVERSITY OF CALIFORNIA BERKELEY · 2023 · $118,247

## Abstract

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 independe...

## Key facts

- **NIH application ID:** 10596613
- **Project number:** 5K99GM143461-02
- **Recipient organization:** UNIVERSITY OF CALIFORNIA BERKELEY
- **Principal Investigator:** Jennifer R Hamilton
- **Activity code:** K99 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $118,247
- **Award type:** 5
- **Project period:** 2022-04-01 → 2023-10-06

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10596613

## Citation

> US National Institutes of Health, RePORTER application 10596613, Uncovering cell-intrinsic restrictions to CRISPR-Cas9 gene editing (5K99GM143461-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10596613. Licensed CC0.

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