A database and cloning system for a genome-wide piRNA interference collection

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Project Description

​In C. elegans, genome-wide tools based on RNA interference (RNAi) have been used to do Systems Biology screens. The collection was based on the observation that bacteria expressing a double-stranded RNA (dsRNA) can elicit an RNAi response in C. elegans if the bacteria are ingested (Timmons and Fire, 1998). This lead to the creation of a genome-wide collection of bacteria expressing dsRNA against most of C. elegans genes (Ahringer and colleagues). This has been used a lot in the field but has some limitations. For example, in the germline, the phenotype from ingested dsRNA is considerably weaker than injected dsRNA. Also, ingested dsRNA is generally inefficient at knocking down genes in the male germline. The germline is of particular interest because this tissue is widely used to study meiosis, early development, gene regulation, inheritance across generations, and stem cell biology. We are developing a novel method to silence genes using molecular machinery from the piRNA pathway (see Ozata et al 2018, Nature Reviews Genetics for an overview) which we are calling piRNA interference (piRNAi). To use this method on a genome-wide scale, we are looking for a motivated student to perform bioinformatics analysis of genome-wide targeting sequences that lead to minimal off-target effects. The project also has a wet lab experimental component, which aims to validate a large-scale cloning method (Golden Gate Cloning) that can be used to generate a genome-wide piRNAi library.​​​
Program - BioScience
Division - Biological and Environmental Sciences and Engineering
Field of Study - ​Genetic engineering and bioinformatics

About the
Researcher

Christian Froekjaer Jensen

Assistant Professor, Bioengineering

Christian Froekjaer Jensen
Professor Froekjaer Jensen's lab studies genome organization and epigenetic gene regulation using C. elegans as a model system. Specifically, our research will focus on three main areas: 

(1) How can non-coding DNA structures prevent epigenetic silencing?
We aim to understand the genetic and mechanistic basis for how a class of non-coding DNA (named PATCs) prevents epigenetic silencing and their interaction with small RNA pathways.

(2) Which gene features enable expression from repressive chromatin?
We will use high-throughput methods to identify: (a) sequence features in enhancers and promoters that tune their expression to their chromatin environment, (b) genomic regions where gene silencing is developmentally regulated, and (c) gene structures (e.g. intron position and content) that regulate expression.

(3) Development of high-throughput genome editing technologies.
We are developing genome editing tools using transposons and Cas9/CRISPR. The long-term goal is to enable genome-scale manipulation for arbitrarily complex re-organization and re-coding of the C. elegans genome. These tools will allow us, and others, to use Synthetic Biology approaches to answer questions about genome structure and function.​

Desired Project Deliverables

​1. A database and web interface for pre-designed piRNAs for C. elegans and other nematode species. 2. A validated cloning method to generate piRNA interference constructs from oligos synthesized on a massively parallel scale.​