# Core C:  Defining regulators of immunity to acute infection using CRISPR screens

> **NIH NIH U19** · HARVARD MEDICAL SCHOOL · 2020 · $148,352

## Abstract

The systematic characterization of gene function, and the understanding of how gene dysfunction leads to
disease states, is one of the largest challenges in biology today. The number of genes multiplied by the
number of cellular states in which they are expressed emphasizes the need for scalable, comprehensive,
unbiased, and robust techniques for perturbing genes to ascertain their functions. CRISPR-Cas9 technology
represents the most promising tool yet described for experimental manipulation of the genome, and a central
focus of Core C is the development, pressure testing, and deployment at-scale of this technology. Core C will
support Research Projects 1 and 2 in this U19 proposal, “Defining regulators of immunity to acute infection
using CRISPR screens.”
Specifically, we will work with the computational core (Core B) and the individual investigators in each of the
two Research Projects to curate a set of target genes for screening in mouse models (Aim 1). We will generate
pooled libraries of gRNA for each project using optimized design algorithms that maximize the efficiency of the
reagents, enabling the use of small numbers of gRNAs per gene and thus the screening of more genes. These
libraries will then be produced as lentivirus for delivery into cells. We will then work with the mouse screening
core (Core D) to determine optimal screening conditions for each of the individual projects (Aim 2). Core D will
execute the in vivo screens and provide genomic DNA samples to Core C for processing and analysis. Core C
will PCR the gRNA inserts, sequence the product via Illumina, and use customized software to deconvolute the
sequencing reads. Finally, Core C will combine information from multiple gRNAs targeting each gene to define
a hit list for each screen, using the STARS algorithm. This hit list is the starting point for systematic network
analysis by Core B. Following primary screens, Core C will support the customized creation of individual
reagents necessary for focused follow-up (Aim 3). This support includes the design and production of
individual gRNA reagents, creation of customized vectors, and implementation of alternative perturbational
technologies, such as CRISPRa, CRISPRi, ORFs, and RNAi. Together, these three aims will accelerate the
research of both Research Projects and enable deep functional knowledge across multiple models of
immunity.

## Key facts

- **NIH application ID:** 9966863
- **Project number:** 5U19AI133524-04
- **Recipient organization:** HARVARD MEDICAL SCHOOL
- **Principal Investigator:** John Gerard Doench
- **Activity code:** U19 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $148,352
- **Award type:** 5
- **Project period:** 2017-07-05 → 2022-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9966863

## Citation

> US National Institutes of Health, RePORTER application 9966863, Core C:  Defining regulators of immunity to acute infection using CRISPR screens (5U19AI133524-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9966863. Licensed CC0.

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