Recently published in a paper on Nature.com in Scientific Reports, Horizon Discovery have conducted a detailed analysis of CRISPR-Cas9 sensitivity (drop-out) screening to come up with a highly improved and optimised platform. In our analysis, we used a custom ultra-complex sgRNA library and capitalised on Horizon’s streamlined screening pipeline to evaluate fundamental aspects of functional genomic screening, including:
- Side-by-side comparison of the impact of a novel tracrRNA sequence on screen performance
- Direct analysis of the efficacy of two different sgRNA design algorithms
- Evaluation of the effect of cell line ploidy on KO rate and screen quality
- Time-resolved gene drop-out analysis to evaluate the kinetics of CRISPR-Cas9 driven gene knockout
Most excitingly, we discovered that a simple adaptation to the tracrRNA sequence can substantially improve the overall rate and kinetics of gene knockout. This modification is expected to increase the stability of the sgRNA-Cas9-DNA complex, and has broad applications for CRISPR function across many applications.
We also found that whilst a third generation library showed improved selection of optimal guides over second generation systems, the most substantive improvements were achieved by adapting the tracrRNA sequence, a modification that essentially equilibrated the performance of the two different library designs.
Finally, our analysis reveals the importance of careful screen design and in understanding the temporal aspects of CRISPR-Cas9 screening as we measured rapid loss of targeted essential genes from the screen population. This effect was even more pronounced in a haploid cell line model, providing a model for high quality and rapid screening platform using Horizon’s eHAP and HAP1 cell line collection.
In summary the findings of this analysis were:
- A simple adaptation to the tracrRNA sequence substantially improves the overall rate and kinetics of gene knockout
- A third generation library showed improved selection of optimal guides over second generation systems but that tracrRNA adaptation equilibrated the performance of two different library designs
- Kinetic analysis revealed rapid effect of gene knock-out by CRISPR-Cas9, profoundly influencing optimal screen design
- Essentiality screening in a hypotriploid cell line yielded robust and powerful hit ID
- Haploid cell line screening provided a modest but significant increase in overall knock-out rates
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