# Assembly of Novel Gene Editing Particles to Understand Genome Surgery in Patient-Derived Cells

> **NIH NIH R35** · UNIVERSITY OF WISCONSIN-MADISON · 2020 · $369,780

## Abstract

There is a fundamental gap in understanding how several components of engineered gene-editing nucleases
achieve gene modification in human cells. Continued existence of this gap represents an important problem
because, until it is filled, use of genome surgery tools will be limited, as it is not clear why various nucleases fail
and why some succeed in producing desired gene edits. The long-term goal is to watch genome surgery in
action to understand the bottlenecks in performing genome surgery on human cells in vitro with precisely
controlled gene-editing particles, comprised of CRISPR-Cas9 components. Particles will be systematically
assembled with various components and delivered in a controlled fashion to patient-derived cells and tissues.
Live, in situ high content imaging and analysis within customized cell substrates will monitor genome surgery.
These capabilities will explore large sequence variation of CRISPR-Cas9 components along with new
assemblies of CRISPR-Cas9 components. The central hypothesis is that new assemblies of CRISPR-Cas9
particles can probe different biological processes of trafficking, DNA-double strand break formation and DNA
repair involved in genome surgery. This hypothesis will be tested with respect to generating two types of gene
edits involving non-homologous end joining (NHEJ) and homology-directed repair (HDR) pathways at several
genomic loci within patient-derived stem cells and tissues. An overarching rationale for the proposed research
programs is that robust gene editing techniques could enable the production of personalized drugs, cell
therapies and gene therapies for future genomic and precision medicine. Guided by strong preliminary data,
this hypothesis will be tested by pursuing three research programs: 1) Assemble Cas9 particles to identify
biological processes that promote "reporter-less" transcript tagging of stem cell fate in culture; 2) Assemble
Cas9 particles to identify biological processes that promote gene correction of diseased mutations in stem
cells; and, 3) Assemble Cas9 particles to identify biological processes that promote gene correction of
diseased mutations in microtissues. Under the first research program, an already proven platform, to assemble
hundreds of unique Cas9 particles and edit patient-derived cells in a multiplexed manner, will be used to
monitor the production of small gene edits by NHEJ within stem cell marker genes. Under the second and third
research programs, this platform will be applied to gene-correct diseased mutations via HDR in induced
pluripotent stem cells and microtissues matured from them. The approach is innovative, in the applicant's
opinion, because it departs from the status quo by systematically changing multiple components at a time
using novel methods in patient-derived cells. The proposed research is significant, because it is expected to
advance and expand understanding of how genome surgery tools can be applied for the generation of
advanced therape...

## Key facts

- **NIH application ID:** 9969447
- **Project number:** 5R35GM119644-05
- **Recipient organization:** UNIVERSITY OF WISCONSIN-MADISON
- **Principal Investigator:** Krishanu Saha
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $369,780
- **Award type:** 5
- **Project period:** 2016-08-19 → 2021-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9969447

## Citation

> US National Institutes of Health, RePORTER application 9969447, Assembly of Novel Gene Editing Particles to Understand Genome Surgery in Patient-Derived Cells (5R35GM119644-05). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9969447. Licensed CC0.

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