Intracellular CRISPR gRNA assembly for massively multiplexed, one pot, (epi)genetic screening

NIH RePORTER · NIH · R21 · $198,360 · view on reporter.nih.gov ↗

Abstract

Project Summary It has been clear for over a decade that cancer is not a single disease and that this heterogeneity is a primary barrier to the understanding of oncogenic mechanisms and treatments. Cancers vary epigenetically and genetically at multiple length and time scales and between patients. There can be many distinct mechanisms driving oncogenic and metastatic processes, even within a single cancerous cell. The challenge researchers and clinicians face is how to understand cancer from the perspective of simultaneous perturbations to multiple genes and proteins. Risk variants identified through genome wide association studies and epiGWAS can be individually perturbed in large experiments comprised of many 384-well plates through RNAi and CRISPR libraries, and even be combinatorially perturbed and screened in `one-pot' using barcoding strategies. However, even state-of-the-art CRISPR screening methods are restricted to functional perturbations of 2 or 3 variants at a time per cell. These restrictions arise from the difficulties repetitive sequences in gRNA arrays present in both expression construct synthesis and stability. Here we propose a new method that will be capable of expressing randomized combinatorial libraries of thousands of distinct gRNAs, with each cell of a population expressing an array of over 30 gRNAs.

Key facts

NIH application ID
9999547
Project number
5R21CA240162-02
Recipient
NORTH CAROLINA STATE UNIVERSITY RALEIGH
Principal Investigator
Albert Keung
Activity code
R21
Funding institute
NIH
Fiscal year
2020
Award amount
$198,360
Award type
5
Project period
2019-09-01 → 2022-08-31