# High-throughput dissection of transcriptional regulation in kidney disease

> **NIH NIH F99** · STANFORD UNIVERSITY · 2020 · $39,452

## Abstract

Chromatin modifications are involved in all basic DNA-templated processes in human cells including
transcription, and are mis-regulated in many diseases. With CRISPR-targeting techniques, we can write
particular chromatin modifications and then measure how gene expression changes in response. This will enable
us to understand how gene expression changes in disease states and how to rationally design therapeutics to
reverse those changes.
 One of the challenges to using CRISPR perturbations to study non-coding regulatory elements is CRISPR off-
target activity. Here, we show that off-target effects in non-coding perturbation experiments can be associated
with significant toxicity in human cells, not only with DNA-cleaving Cas9, but also with epigenome-modifying
CRISPRi/a tools. After removing off-target-prone guide RNAs, we can use CRISPR to accurately link non-coding
regulatory elements with genes.
 These perturbation experiments are critical to learn the causal functions of chromatin modifications at
specific genomic elements. However, most experiments to date have used CRISPRi, with one particular KRAB
domain that establishes one particular heterochromatin state. Existing tools to manipulate chromatin state are
largely drawn from a small fraction of the thousands of natural chromatin regulatory complexes; most suffer from
partial or transient effects, and exhibit high variability across loci and cell types. A more complete toolbox of
compact, efficient domains for pathway-specific chromatin perturbations will transform our ability to determine
the causal function of particular chromatin modifications across the human genome. Here, we propose to
systematically measure the gene expression effects of recruiting chromatin regulator protein domains to a
promoter. This is made possible by our recent development of a high-throughput chromatin regulator recruitment
assay in human cells, capable of measuring activity for tens of thousands of regulator domains simultaneously.
Using this system, we will recruit-and-release CR variants from the promoter, and then measure the magnitude
and permanence of transcriptional silencing at a reporter locus. We will then use epigenomic mapping assays to
determine the chromatin modifications that underpin the silencing functions of these novel chromatin regulators.
After characterizing thousands of domains drawn from all the different chromatin regulatory complexes, we will
create and share a detailed resource of compact and efficient domains that can be fused onto CRISPR DNA-
binding proteins in order to recruit desired chromatin regulatory complexes to act upon a genomic element.
 In order to positively impact human health with these approaches, I propose to leverage this training to
develop new methods that dissect transcriptional dysregulation in kidney disease during the postdoctoral phase.

## Key facts

- **NIH application ID:** 10059105
- **Project number:** 1F99DK126120-01
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** Josh Tycko
- **Activity code:** F99 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $39,452
- **Award type:** 1
- **Project period:** 2020-09-01 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10059105

## Citation

> US National Institutes of Health, RePORTER application 10059105, High-throughput dissection of transcriptional regulation in kidney disease (1F99DK126120-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10059105. Licensed CC0.

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