A summary of a published arrayed CRISPR-Cas9 screen with synthetic crRNAs targeting ubiquitin enzymes

Functional genomics screening with an Edit-R crRNA library

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!

Pros and cons of different CRISPR-Cas9 reagents for screening

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.

Edit-R Synthetic crRNA:tracrRNA works very well for arrayed screening

High levels of editing efficiency by a GMNN-targeting positive control crRNA are demonstrated by the following findings:

  • Significant number of cells exhibit large nuclear size phenotype
  • Significant reduction of protein expression by immune fluorescence
  • > 80% of targeted DNA edited, as evaluated by sequencing
  • crRNA:tracrRNA knockout phenotype comparable to siRNA knockdown phenotype

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.

  • Control crRNA had z'-factors ≥ 0.5, demonstrating a high-quality screen
  • crRNA:tracrRNA screens were highly reproducible
  • Multiple crRNAs targeting the same gene show higher correlation than that seen for multiple siRNAs. Likely due to fewer off-targets with CRISPR-Cas9 compared to RNAi.
  • crRNA:tracrRNA identified the same genes as hits that were identified by siRNA. However, overall crRNA:tracrRNA identified more significant genes than siRNA

Comparison of screening reagents

Synthetic crRNA:tracrRNA


Requires source of Cas9 & crRNA:tracrRNA

Requires siRNA only

Differences in phenotypic strength due to cleavage/repair mechanism

Differences in phenotypic strength due to targeting mRNA

High specificity with Edit-R predesigned crRNA

Off-target effects by microRNA-like seed-mediated effects

Multiple crRNA sequences result in phenotype

Multiple siRNA sequences result in phenotype

Consistent phenotype among crRNAs for greater statistical significance

More variability among siRNAs targeting the same gene

crRNA confirms siRNA hits

siRNA confirms crRNA hits

Discussion topics

  • High-throughput, genome-wide screening is feasible with Edit-R arrayed crRNA libraries
  • Phenotypes for essential genes may be observed with synthetic crRNA:tracrRNA screens while being lost in pooled sgRNA screens
  • Cas9 expression levels can affect the penetrance of the phenotype
  • Effective guide RNA design is important and warrants more investigation in the future


  1. Tan J, Martin SE (2016) Validation of Synthetic CRISPR Reagents as a Tool for Arrayed Functional Genomic Screening. PLoS ONE 11(12): e0168968. doi:10.1371/journal.pone.0168968

Additional Resources

Edit-R crRNA Library
  • Arrayed collections of predesigned synthetic crRNA for high-throughput screening across entire gene families for human and mouse.
Cherry-pick Library Plater
  • Customize and order plates of predesigned crRNA for knockout studies for your targets of interest.
Arrayed synthetic CRISPR RNA screen in primary T-cells validates HIV host factors - Featured Article
  • Synthetic crRNA and tracrRNA can be used in high-throughput screening applications
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  • Find out if synthetic crRNA in multiwell plates is the right option for your CRISPR-Cas9 project
Increase the throughput and reproducibility of your CRISPR-Cas9 screen - Featured Article
  • Reverse transfection for high-throughput CRISPR studies