Generation and Validation of a Novel Genome-Scale Inducible RNAi Library for Functional Genetics in Leishmania braziliensis.

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Project Summary Leishmania parasites cause a suite of devastating Neglected Tropical Diseases that afflict as many as one million people per year. Genetic tools for understanding important questions in Leishmania biology are currently quite limited. Most Leishmania species lack the capacity for RNA interference (RNAi), precluding the implementation of genome-scale RNAi library screens like those that have revolutionized molecular genetics in the related kinetoplastid T. brucei. However, the Viannia subgenus, which includes Leishmania braziliensis, has retained the RNAi machinery. We have developed the first inducible RNAi system for L. braziliensis and have adapted it for high throughput Loss-of-Function RNAi screens. The overall goal of this proposal is to generate and validate a novel, genome-scale, inducible RNAi library in L. braziliensis that will serve as a versatile new genetic tool for the field. Despite differences in the capacity for RNAi, the genome organization, life cycle stages, and disease pathology of L. braziliensis are very similar to other Leishmania species, making it likely that results from L. braziliensis RNAi screens will be broadly applicable. Our inducible expression system is based on conditional site-specific recombination by a split-Cre recombinase (DiCre) that is only active in the presence of the inducer rapamycin (Rap). For inducible RNAi, Rap treatment activates DiCre to invert a pair of tandemly duplicated RNAi target sequences into an antiparallel orientation. Transcription of this inverted repeat generates a long double-stranded RNA stem-loop that serves as a trigger for RNAi knockdown of the targeted mRNA. Specific Aim 1 has three components: 1) We will generate a genome-scale plasmid library of random ~1 kb L. braziliensis genomic DNA fragments that will serve as RNAi targeting sequences. 2) This plasmid library will be converted to a barcoded inducible stem- loop RNAi library via a method developed in our lab called Strand Displacement Duplication (SDD); 3) The inducible stem-loop RNAi library will be stably transfected into a DiCre-expressing L. braziliensis strain, using CRISPR/Cas9 cleavage to improve library integration efficiency. Because the overall goal is to make the RNAi library available as a resource for the community, we will validate the library by performing proof-of-principle screens to identify drug resistance genes (Gain-of-Fitness) and pathways required for normal growth (Loss-of- Fitness) (Specific Aim2). The representation of RNAi target sequences in library under various growth conditions will be assessed via an adaptation of the barcode sequencing method (Bar-seq). The L. braziliensis RNAi library platform will provide an innovative and much-needed new tool for high throughput genetic screens in Leishmania. We anticipate that genome-scale RNAi screens 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.