# Recombinase-free conditional knock-in through alternative splicing

> **NIH NIH R21** · WEST VIRGINIA UNIVERSITY · 2024 · $228,000

## Abstract

Project Summary
This project will develop a new tool, SpliceTag, for conditional knock-in/knockout and demonstrate its utility.
SpliceTag uses highly cell type specific alternative exons as a vehicle for carrying protein and RNA tags for
conditional labeling, or premature stop codons for protein knockout. It can be introduced in an intron of the
target gene through CRISPR/Cas9 induced homology directed repair. The SpliceTag exon will then be spliced
in mRNA labeling the protein in the desired cell type. In Aim 1 we will create a mouse line in which we use a
SpliceTag in Rpl22 to label ribosomes with ALFA peptide in photoreceptor cells. The photoreceptor specific
SpliceTag is based on Ttc8 microexon 2a which we have shown in the past to be highly specific to
photoreceptor cells. To demonstrate the utility of our approach we will use the Rpl22 SpliceTag line to analyze
translational efficiency in photoreceptor cells and how translation is controlled during the diurnal cycle. In Aim 2
we will determine the feasibility of using SpliceTag to label proteins in epithelial cell and inner retina neurons.
We will also determine if it can incorporate commonly used tags, SNAP and CBP/Strep, that are significantly
larger than the Ttc8 exon 2a on which SpliceTag is based. Since SpliceTag does not rely on recombinases it
can enable experimental designs where recombinase and floxed alleles are used for genetic manipulations in
parallel with SpliceTag, including knockouts in cell types other than the cell type in which the protein is being
labeled.

## Key facts

- **NIH application ID:** 10866802
- **Project number:** 1R21EY036144-01
- **Recipient organization:** WEST VIRGINIA UNIVERSITY
- **Principal Investigator:** Peter Stoilov
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $228,000
- **Award type:** 1
- **Project period:** 2024-06-01 → 2026-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10866802

## Citation

> US National Institutes of Health, RePORTER application 10866802, Recombinase-free conditional knock-in through alternative splicing (1R21EY036144-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10866802. Licensed CC0.

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