siRNA Screening: Development of Hit Stratification Strategies
Poster Mar 10, 2015
Žaklina Strezoska, Annaleen Vermeulen, Emily M. Anderson, Anja Smith, Devin Leake
While synthetic siRNA libraries are powerful tools for functional genomic screens, off-target effects mediated by siRNA seed interactions with the 3' UTR of unintended targets can result in false positives. Given the frequency of off-target effects in some assays, the development of hit validation/stratification strategies is imperative. In the following study we have compared two strategies for identification of high confidence hits: 1) a multiple reagent approach where two or more individual siRNAs induce the same phenotype and 2) a chemical modification approach where hit confirmation is achieved using pools of siRNA that contain specificity enhancing modifications. A comparison of these two strategies (using a collection of primary hits generated from a cell viability screen) reveals significant overlap between the high confidence hits identified. However, for low confidence hits, i.e. where a single siRNA induces a phenotype, the concern is that an important hit will be missed. To determine if the phenotype is due to gene targeting or a seed-mediated off-target effect, a chimeric approach was used whereby a gene-specific seed sequence is introduced into a non-targeting siRNA scaffold. Together, these data provide well-defined approaches for prioritization of hits derived from RNAi screens.
Psychiatric Risk Gene Cacna1c and Early Life Stress: Potential Gene-Environment interactions?Poster
Early life stress (ELS) is highly associated with development of psychopathology
and mood disorders in adulthood. Genetic studies have identified variation in the gene calcium voltage-gated channel subunit alpha1C (CACNA1C) to increase risk for several psychiatric disorders. This poster assessed the expression of Cacna1c following prepubertal stress.
A new method for generating arrayed RNAi screening tools for any organismPoster
RNA interference (RNAi) using small interfering RNAs (siRNAs) is an important technology for down-regulation of gene expression and a powerful tool to study cellular processes and pathways. Previously, large collections of siRNAs were available only for traditional experimental model systems, such as human and mouse, and predominantly provided as chemically synthesized libraries.READ MORE
Knockout of microRNAs Using the CRISPR-Cas9 System with Paired Synthetic crRNAsPoster
We utilized paired synthetic crRNAs coupled with our synthetic tracrRNA in cells transduced with lentiviral Cas9 to perform a functional knockout on hsa-miR-221. This three-part system (crRNA, tracrRNA and Cas9) has demonstrated efficient gene editing when used with only one guide RNA, but the goal was to use two crRNAs to remove the entire stem-loop.READ MORE