# Generation and validation of a novel inducible overexpression library for genome-scale genetic screens in Leishmania

> **NIH NIH R21** · OREGON HEALTH & SCIENCE UNIVERSITY · 2024 · $35,035

## Abstract

Project Summary
 Leishmania parasites cause a suite of devastating Neglected Tropical Diseases that afflict as many as
one million people per year. At least 20,000 are killed each year by the deadly visceral form of the disease,
which is caused by L. donovani. Genetic tools for understanding important questions in Leishmania biology are
currently quite limited. Most Leishmania species lack the capacity for RNA interference (RNAi), which
precludes the implementation of genome-scale RNAi library screens like those that have revolutionized
molecular genetics in the related kinetoplastid T. brucei. As is the case for knocking down protein expression
via RNAi, expressing proteins at higher levels than normal (i.e., overexpression) can confer resistance to drugs
or otherwise enhance survival (Gain-of-Fitness = GnFt), or cause deleterious effects on cell fitness (Loss-of-
Fitness = LsFt) that can reveal important pathways and protein functions. Genome-scale overexpression
libraries have proven valuable for a wide range of genetic screens in multiple species, including T. brucei. The
goal of this proposal is to generate and validate a novel, genome-scale, inducible overexpression library in
Leishmania that will serve as a versatile and broadly applicable new genetic tool for the field.
 We propose to generate a library encoding the majority of L. donovani proteins (~7500 Open Reading
Frames, or LdORFs). PCR amplified LdORFs will be directionally cloned into Gateway Entry vectors to
facilitate transfer of the library to any Gateway compatible vector (Aim 1), resulting in a LdORFeome plasmid
library. The LdORFeome will be transferred into novel inducible Leishmania expression vectors developed in
our lab (Aim 1). The resulting library will be stably transfected into Leishmania to generate an inducible
Leishmania donovani ORFeome overexpression library (the LdOX library). Because the overall goal is to
make the LdOX library available as a resource for the community, we will validate the library by performing
proof-of-principle overexpression screens to identify drug resistance genes (GnFt) and pathways required for
normal growth (LsFt) (Aim2).
 The LdOX library will provide an innovative and much-needed new tool for high throughput genetic
screens in Leishmania. We anticipate that screens with the LdOX library will have a sustained impact on the
field by revealing mechanisms of drug action and resistance, and uncovering proteins and pathways required
for surviving any growth condition or lifecycle stage.

## Key facts

- **NIH application ID:** 10885443
- **Project number:** 3R21AI166002-02S1
- **Recipient organization:** OREGON HEALTH & SCIENCE UNIVERSITY
- **Principal Investigator:** PHILLIP A YATES
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $35,035
- **Award type:** 3
- **Project period:** 2023-02-13 → 2025-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10885443

## Citation

> US National Institutes of Health, RePORTER application 10885443, Generation and validation of a novel inducible overexpression library for genome-scale genetic screens in Leishmania (3R21AI166002-02S1). Retrieved via AI Analytics 2026-06-12 from https://api.ai-analytics.org/grant/nih/10885443. Licensed CC0.

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