# Development of potent and predictable Cas9 gene activation tools through high-throughput screening

> **NIH NIH R01** · BRIGHAM AND WOMEN'S HOSPITAL · 2024 · $10,025

## Abstract

Project Summary
The ability to manipulate the expression of genes in cells and organisms is foundational to the
study of genetics. The CRISPR-Cas9 genome editing toolkit has revolutionized our ability to
modify the genome and epigenome precisely. While CRISPR tools have been optimized to allow
for robust, tunable, and predictable repression and inactivation of gene expression, approaches
to induce gene expression (CRISPR activation, or CRISPRa) are less robust and reproducible.
Through a combination of innovative high-throughput screens and cutting-edge computational
modeling, we will develop a cohort of simple, robust, tunable, and predictable CRISPR-based
tools to increase the expression of any mouse or human gene.
We have developed a high-throughput sequencing-based assay system, Self-sustaining Peptide
Activator Reporter-seq (SPARq), which enables quantitative screening of thousands of
candidate gene activating peptides and combinations thereof to monitor and optimize their gene
activation strength. In Aim 1, we will iteratively employ SPARq to systematically evaluate and
optimize multiple features of gene activating peptides, including activation peptide identity,
combination, linker, and CRISPRa method. We will perform SPARq screens in distinct cell types
and with distinct promoter architectures to identify tools that work consistently, designing a set of
CRISPRa tools that are significantly more potent and consistent than the current state-of-the-
art.
In Aim 2, we will improve the predictability and consequently the utility of CRISPRa through a
novel high-throughput reporter assay and a computational effort to model the features
associated with CRISPRa potency. We have designed an approach, CRISPR Outcome and
Phenotype screening, that combines a sensitive reporter assay with a native genomic
phenotypic measurement to profile the activity of a CRISPRa tool at thousands of target sites.
Using data collected through this pipeline, we will develop an algorithm that takes as input one
of the CRISPRa tools developed in Aim 1, a cell type, gene, and CRISPR guide RNA and
outputs an accurate estimate of the expression of that gene following CRISPRa treatment. We
will validate the accuracy of this algorithm to enable tunable gene activation over an extensive
dynamic range in human HepG2 and K562 cells, providing it to the genetics community as a
webtool.
Altogether, the efforts described in this proposal will pioneer a next generation toolkit to enable
more robust gain-of-function genetic manipulation and screening.

## Key facts

- **NIH application ID:** 11093039
- **Project number:** 3R01GM143249-03S1
- **Recipient organization:** BRIGHAM AND WOMEN'S HOSPITAL
- **Principal Investigator:** Richard I Sherwood
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $10,025
- **Award type:** 3
- **Project period:** 2022-08-01 → 2025-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11093039

## Citation

> US National Institutes of Health, RePORTER application 11093039, Development of potent and predictable Cas9 gene activation tools through high-throughput screening (3R01GM143249-03S1). Retrieved via AI Analytics 2026-06-12 from https://api.ai-analytics.org/grant/nih/11093039. Licensed CC0.

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