A functional genomics tool for high throughput cell free analysis of regulatory components

Project Summary Here we seek to develop a novel functional genomics tool a RNA Polymerase II dependent RNAPII eukaryotic cell free expression system that allows for rapid characterization of human regulatory components There is a pressing need for new tools that can analyze the exponentially increasing amount of genomic data that has been collected through NHGRI sequencing initiatives and through advances in next generation sequencing technology Hidden within this genomic data is understanding to causes of human pathology Understanding genomic data will become increasingly critical as new initiatives such as TOPMed seek to tie primary data to clinical outcomes Synvitrobio s technology produces cell free systems that are able to express multiple DNA sequences without needing to grow cells This allows for x higher throughput analysis at x the time and cost of equivalent cellular expression The cell free system effectively prototypes a cell They can be thought of as simplified and open environments to engineer biological complexity while conserving the native cellular milieu of the originating source The system takes in single or multiple DNA s and conducts transcription and translation to produce functional protein We propose developing a RNAPII dependent eukaryotic cell free expression system that is able to prototype a eukaryotic cell line Current eukaryotic cell free systems are able to conduct either native transcription or separately native translation but not both simultaneously Both native transcription and native translation are necessary for the end system to be capable of high throughput functional genomics exploration In the proposed Phase I feasibility effort we will first demonstrate a HeLa combined nuclear and cytoplasmic cell free expression system supporting RNAPII dependent native transcription and native translation Aim We will also demonstrate two examples of gene regulation in a eukaryotic cell free system a RNAPII independent example of operator repressor binding and a RNAPII dependent example of allele specific gene regulation Aim The long term Phase II goal is to demonstrate the system s ability to rapidly screen genomic data going from bioinformatics collection DNA synthesis expression and assay in a day with a demonstration that collected results mirror expected results from cellular experiments Project Narrative A functional genomics tool from eukaryotic cell free systems benefits the public by providing a platform that can rapidly screen and develop functional conclusions from the increasing amount of collected personal and human genomics data This aids in identifying fundamental underlying mechanisms of associated genetic variants and related causes of human pathology Understanding genomics data will be critical to developing personalized disease cures