# shRNAmir and CRISPR sgRNA Library Construction Core

> **NIH NIH P01** · UNIVERSITY OF FLORIDA · 2022 · $140,145

## Abstract

PROJECT SUMMARY / ABSTRACT
Core B (Pipkin)
Short hairpin RNAs in microRNA contexts (shRNAmirs) and CRISPR-Cas9 guide RNA (sgRNA) are powerful
tools for experimental RNA interference (RNAi) and gene-disruption in the immune system, respectively.
However, virtually all current approaches require pre-activation of naive cells to render them susceptible for gene
transfer, typically with retroviral or lentiviral vectors, and cause constitutive RNAi or immediate gene-disruption.
These issues complicate studying genes that function prior to retroviral delivery, and clarifying the temporal
requirements of genes at different stages of immune responses. In Core B, we describe development of multiple
innovative tools and methods that substantially expands the utility of shRNAmirs and sgRNAs to study gene
function. We demonstrate generation of unmanipulated naive CD4 and CD8 T cells carrying conditionally
regulated retroviral constructs that enable conducting inducible and reversible RNAi in vivo, and extend this
system to a novel approach that facilitates large in vivo pooled screens using inducible shRNAmirs in untouched
naive cells. We also demonstrate CRISPR-Cas9 guide RNA (sgRNA) gene disruption in primary B and T cells,
and its use during in vivo immune responses to analyze B and T cell function. The combination of these
shRNAmir and sgRNA approaches are synergistic and constitute an innovative and rigorous experimental
platform for rapidly dissecting gene function in T and B cells during in vivo immune responses. Core B builds on
multiple existing libraries of high-fidelity shRNAmir clones, which include those that target all genes encoding
mammalian chromatin regulator factors (312 genes, ~1,700 shRNAmirs), and all mammalian transcription factors
(2,083 genes, ~8,000 shRNAmirs). The overarching goal of Core B is to provide essential tools for executing the
proposed in vivo loss-of-function studies proposed in Projects 1-3 of this P01. Our specific objectives are to sub-
clone pooled shRNAmir libraries using an inducible vector for conditional pooled screens in naive T cells that
are proposed in Projects 2 and 3 (Aim 1); to produce high-quality, ready-to-transfect retroviral plasmid DNA
from cloned libraries of shRNAmirs and sgRNAs for Projects 1-3 (Aim 2); and to re-apply existing arrayed
shRNAmir libraries and build custom shRNAmir and gRNA clone sets in appropriate vectors based on gene
targets prioritized during the evolution of Projects 1-3 to facilitate detailed follow-up studies (Aim 3).

## Key facts

- **NIH application ID:** 10488582
- **Project number:** 5P01AI145815-04
- **Recipient organization:** UNIVERSITY OF FLORIDA
- **Principal Investigator:** Matthew Eugene Pipkin
- **Activity code:** P01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $140,145
- **Award type:** 5
- **Project period:** 2020-08-01 → 2025-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10488582

## Citation

> US National Institutes of Health, RePORTER application 10488582, shRNAmir and CRISPR sgRNA Library Construction Core (5P01AI145815-04). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10488582. Licensed CC0.

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