Error message

  • Deprecated function: Optional parameter $feed_nid declared before required parameter $context is implicitly treated as a required parameter in include_once() (line 1439 of /local/drupal/imat/includes/bootstrap.inc).
  • Deprecated function: Optional parameter $input declared before required parameter $form_state is implicitly treated as a required parameter in require_once() (line 12 of /local/drupal/imat/sites/all/modules/media/media.module).
×

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


Year of Award:
2019
Status:
Active
Award Type:
R21
Project Number:
CA240162
RFA Number:
RFA-CA-18-002
Technology Track:
Molecular & Cellular Analysis Technologies
PI/Project Leader:
KEUNG, ALBERT
Other PI or Project Leader:
Not Applicable
Institution:
NORTH CAROLINA STATE UNIVERSITY RALEIGH
Project SummaryIt has been clear for over a decade that cancer is not a single disease and that this heterogeneity is a primarybarrier to the understanding of oncogenic mechanisms and treatments. Cancers vary epigenetically andgenetically at multiple length and time scales and between patients. There can be many distinct mechanismsdriving oncogenic and metastatic processes, even within a single cancerous cell. The challenge researchers andclinicians face is how to understand cancer from the perspective of simultaneous perturbations to multiplegenes and proteins. Risk variants identified through genome wide association studies and epiGWAS can beindividually perturbed in large experiments comprised of many 384-well plates through RNAi and CRISPRlibraries, 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 3variants at a time per cell. These restrictions arise from the difficulties repetitive sequences in gRNA arrayspresent in both expression construct synthesis and stability. Here we propose a new method that will becapable of expressing randomized combinatorial libraries of thousands of distinct gRNAs, with each cell of apopulation expressing an array of over 30 gRNAs.