• High-throughput functional genomics screen using an Edit-R crRNA library

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

    High-throughput functional genomics screen using an Edit-R crRNA library

    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 siRNA
    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

    References

    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

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