AN OPTIMIZED DESIGN FOR SINGLE COPY SHORT HAIRPIN RNAI


Year of Award:
2015
Award Type:
R21
Project Number:
CA196379
RFA Number:
RFA-CA-14-003
Technology Track:
Molecular & Cellular Analysis Technologies
PI/Project Leader:
HAMMOND, SCOTT M
Other PI or Project Leader:
N/A
Institution:
UNIV OF NORTH CAROLINA CHAPEL HILL
Short hairpin RNA (shRNA) mediated RNAi is a well-established method for investigating cancer pathways and is a promising future therapeutic strategy. In particular, delivery of shRNAs by retroviral transduction enables RNAi knockdown in mouse models and cell lines refractory to transfection. The primary disadvantage of retroviral shRNA-based experiments is the reduced target gene knockdown frequently observed when a single copy of the shRNA vector is integrated. We have investigated the fate of shRNA precursors using a novel deep sequencing strategy we have previously developed. We find over 99 percent of precursor molecules are diverted to a non-productive biogenesis route, severely limiting the amount of mature RNA produced. In this proposed application we describe a screening strategy to optimize shRNA design and maximize target gene knockdown potential. Our long term goal, beyond the scope of this grant application, is to construct genome-wide shRNA libraries using our optimized shRNA design.