Since the completion of the Human Genome Project, a major effort in the biomedical sciences has been to understand the function of proteins using perturbation. Functional screening platforms based on RNAi and CRISPR, in particular, have been instrumental in pursuing this goal. Yet despite their wide use and extraordinary strengths, even todays state-of-the-art platforms are still limited in their capabilities. Two prominent limitations include (1) The inability to study the function of genes that are essential for cellular growth, and (2) the slow dynamics of protein depletion that enable adaptation mechanisms that can influence the results. To overcome these limitations, I propose to develop a novel pooled screening platform based on rapid and direct modulation of proteins in human cells at a genome-scale. This platform will be based on multiplexed endogenous tagging of genes with HaloTag, a bioorthogonal protein domain capable of binding a variety of small molecule ligands that can be used to either increase or decrease the stability of the fusion protein. In this proposal I will optimize pooled multiplexed endogenous gene tagging using a unique intron targeting approach which our lab has developed. Genome-scale HaloTag fusion libraries will be used to screen for effects of acute protein degradation and destabilization, identify genes that are prone for transcriptional adaptation, and identify aggregating proteins in an unbiased manner using a different fluorescence ligands.

Public Health Relevance

Genome-wide perturbation screening approaches are at the root of many of the most important biological discoveries. Existing screening tools affect protein levels indirectly by targeting the genome or the transcriptome which makes them prone to compensatory mechanisms and loss of growth essential genes. Here I describe the development of a screening method in which proteins are directly and rapidly targeted at a genome scale.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
NIH Director’s New Innovator Awards (DP2)
Project #
1DP2GM137416-01
Application #
9780773
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Mcguirl, Michele
Project Start
2019-09-30
Project End
2024-05-31
Budget Start
2019-09-30
Budget End
2024-05-31
Support Year
1
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Children's Hospital of Philadelphia
Department
Type
DUNS #
073757627
City
Philadelphia
State
PA
Country
United States
Zip Code
19146