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A summary of a published arrayed CRISPR-Cas9 screen with synthetic crRNAs targeting ubiquitin enzymes
An article published in PLoS ONE by Jenille Tan and Scott E. Martin1 is packed with great information on how these screeners from Genentech validated the use of arrayed Edit-R synthetic crRNA libraries for their discovery work. Keep reading for a summary of their findings!
Pooled lentiviral sgRNA libraries are most commonly used for genome-scale screens to date, and have shown the power of CRISPR-Cas9-mediated knockout screening. While this approach does not require automation, the logistics of transduction and maintenance of large numbers of cells combined with requirement for next-generation sequencing can be onerous. In addition, this technology can only be used in assays where constructs are enriched or depleted, for example, using viability, cell sorting or synthetic lethal assays.
Arrayed, one-gene-per-well screening is applicable to a much wider variety of assays including enzymatic endpoint or sophisticated high-content imaging assays. Arrayed lentiviral sgRNA constructs are challenging to apply in arrayed format. The main challenge of the technology is generation of lentiviral particles for hundreds or thousands of wells with reproducible and sufficiently high titer while complying with safety regulations. In addition, lentiviral sgRNA transduction, antibiotic selection, and expression often requires longer time points, which can require splitting of cells. This can be difficult to scale to large number of genes.
This publication investigates the feasibility of using a two-part synthetic guide RNA (Edit-R crRNA and tracrRNA) in a high-throughput arrayed screen. The authors reverse transfected synthetic crRNA:tracrRNA into a Cas9-expressing HCT-116 cell line, and a phenotypic assay for aberrant DNA replication was performed.
High levels of editing efficiency by a GMNN-targeting positive control crRNA are demonstrated by the following findings:
A screen was performed for aberrant DNA replication with the Dharmacon Edit-R™ Human Ubiquitin Enzymes crRNA library (640 genes; 4 crRNA per gene) and compared the results to an ON-TARGETplus siRNA library targeting the same genes.
Arrayed collections of predesigned synthetic crRNA for high-throughput screening across entire gene families for human and mouse.
Customize and order plates of predesigned crRNA for knockout studies for your targets of interest.
Synthetic crRNA and tracrRNA can be used in high-throughput screening applications
Find out if synthetic crRNA in multiwell plates is the right option for your CRISPR-Cas9 project
Reverse transfection for high-throughput CRISPR studies