# Mosaic in Vivo Perturbation (MVP) for systematic assessment of gene function in the eye

> **NIH NIH R01** · HARVARD MEDICAL SCHOOL · 2024 · $693,571

## Abstract

Project Summary
Genetic screens have been a mainstay of scientific research in animals for over a century contributing
immensely to our understanding of molecular cell biology, animal development, and numerous diseases
including cancer and neurodegeneration. Despite their success, genetic screens have been limited by several
factors. Pleiotropy, when the same gene is used in more than one process, can mask gene function in one
tissue if the same gene is required at an earlier stage. Redundancy, when more than one gene is sufficient for
a given function, can prevent the identification of genes with homologues with overlapping expression patterns.
Pleiotropy and redundancy are both quite common in animals precluding the functional assessment of many
genes. And finally, traditional genetic screens are very labor intensive, especially in vertebrates such as
zebrafish and mice, which has limited their full use. In this multi-PI grant, we will combine the expertise of Dr.
Allon Klein in technology development and computation for single cell approaches with that of Dr. Sean
Megason on zebrafish genetics and developmental biology. Here we propose a novel genetic screening
approach termed Mosaic in Vivo Perturbation (MVP) that can overcome the difficulties of pleiotropy,
redundancy, and scalability. MVP relies on: i) recent technological advances including single-cell RNAseq,
CRISPR/Cas9 pooled perturbations; and ii) the remarkable regulative ability of animals especially fish and
mammals to undergo “mosaic complementation”–where wildtype cells fill the niche of mutant cells in genetic
mosaics allowing for the formation of phenotypically normal organisms. Here we propose the development of
CRISPR-based MVP libraries using transposon based expression of multiple sgRNAs in zebrafish that will
allow for hundreds of cells per embryo to each receive a different defined mutation or combination of mutations
for putatively redundant genes. Given the regulative nature of zebrafish, these embryos will most often be
phenotypically normal; however, the proportion of cells with a given genotype that form each cell type will
depend on the fitness of each cell's genotype for that cell type (if a gene is important for forming a cell type, its
sgRNA will be underrepresented). Using scRNA-Seq we will jointly genotype (determine sgRNAs) and
phenotype (determine cell type) large numbers of cells from MVP zebrafish embryos to quantify the function of
all genes in the library for all measured cell types. Here we focus on the eye, because of its large size and
dissectability in zebrafish, remarkable development, and the large unmet medical challenges of eye diseases.
In three largely independent aims, we will 1) optimize the MVP approach through iterative testing culminating in
measuring the function of the complete TFome in the eye, 2) develop an ultra-scalable readout approach for
cell type based on our novel “capsule” technology, and 3) develop validated whole genome MVP CRI...

## Key facts

- **NIH application ID:** 10940839
- **Project number:** 1R01EY036381-01
- **Recipient organization:** HARVARD MEDICAL SCHOOL
- **Principal Investigator:** Allon Moshe Klein
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $693,571
- **Award type:** 1
- **Project period:** 2024-09-01 → 2029-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10940839

## Citation

> US National Institutes of Health, RePORTER application 10940839, Mosaic in Vivo Perturbation (MVP) for systematic assessment of gene function in the eye (1R01EY036381-01). Retrieved via AI Analytics 2026-08-01 from https://api.ai-analytics.org/grant/nih/10940839. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*