A workflow for CRISPR-Cas9 high throughput arrayed screening with synthetic crRNA

DATE:  June 23, 2016
TIME:  8am Pacific time, 11am Eastern time

• Key factors in successful arrayed crRNA screening
• Benefits of the dual CRISPR guide RNA system
• Guidelines for optimization of reverse transfection with crRNA and tracrRNA
• Recommendations for Cas9 nuclease selection in screening applications
• Availability of supporting products and resources

There is rapidly growing interest in using the CRISPR-Cas9 system for functional screening, both as a primary screening tool and as an orthogonal tool for RNAi hit validation. High throughput synthesis, combined with a proprietary algorithm that selects highly functional and specific guide RNAs, allows rapid generation of CRISPR RNA (crRNA) collections in arrayed formats. Arrayed crRNA screens offer the advantage of applying sophisticated assays, such as those requiring high-content microscopy, to investigate phenotypes that involve intracellular localization, morphological changes, or require time-lapse investigation. While pooled lentiviral sgRNA screens have demonstrated utility for loss-of-function studies, the types of assays used are typically limited to viability, sensitization, and some reporter assays. We will demonstrate the application of arrayed screening with synthetic crRNA libraries across multiple assay types, and present considerations for experimental success.