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

> **NIH NIH R35** · UNIVERSITY OF WISCONSIN-MADISON · 2022 · $415,684

## Abstract

PROJECT SUMMARY/ ABSTRACT. There continues to be a fundamental gap in understanding how CRISPR-
based genome editors produce gene modifications in different human cells. A lack of understanding of why
various editors fail and why some succeed in creating desired gene edits - while retaining full cell and tissue
functionality - limits the use of genome editing tools. By observing genome editing in real-time within patient-
derived cells in vitro, I seek to understand the bottlenecks in performing genome editing on human cells with
precisely-controlled genome editor particles. Particles will be systematically assembled with various DNA, RNA,
and polymeric components and delivered to patient-derived cells and microtissues. In situ high content imaging
and analysis within customized cell substrates will monitor genome editing at multiple scales. 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 the genome editing of human cells
and tissues, as well as downstream effects on biological processes involving cell cycle arrest and
morphogenesis. This hypothesis will be tested within patient-derived stem cells and tissues for both gene
disruption and correction. An overarching rationale for the proposed research is that an improved understanding
of fundamental biological processes involved with genome editing could enable the development of novel cell
therapies and gene therapies for future genomic and precision medicine. Guided by strong productivity in the
current early stage R35 award, I will pursue three research programs: 1) Assemble Cas9 particles to identify
chromatin structures within human cells that promote gene correction; 2) Assemble Cas9 particles to identify
delivery and DNA repair processes that promote gene correction within stem cells; and, 3) Assemble Cas9
particles to identify cell proliferative and tissue morphogenesis processes that promote gene correction of
diseased mutations in patient-derived microtissues. Under the first research program, editing will occur at target
genes that have variable chromatin structures within induced pluripotent stem cells (iPSCs), differentiated
progeny, and with small-molecule treatment. Under the second and third research programs, genome editors
will be applied to gene-correct diseased mutations in iPSCs, 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 our understanding of how genome editing tools can be applied
for the generation of advanced therapeutics, ranging from targeted small molecules to cell/tissue therapies.
Ultimately, such knowledge would solidify the foundation for new translational proj...

## Key facts

- **NIH application ID:** 10410499
- **Project number:** 5R35GM119644-07
- **Recipient organization:** UNIVERSITY OF WISCONSIN-MADISON
- **Principal Investigator:** Krishanu Saha
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $415,684
- **Award type:** 5
- **Project period:** 2016-08-19 → 2026-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10410499

## Citation

> US National Institutes of Health, RePORTER application 10410499, Assembly of Novel Gene Editing Particles to Understand Genome Surgery in Patient-Derived Cells (5R35GM119644-07). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10410499. Licensed CC0.

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