Hi,
Can I ask what the sgRNA level log fold change score is in Sanger CRISPR (Project Score, CERES)? sgRNA is a part of CRISPR, why sgRNA will have quantity change during CRISPR study? Why is it important to know lof fold change?
Thank you!
Hello! Log-fold change (LFC) is a useful metric for quantifying cell fitness effects in CRISPR knockout experiments like Project Score and Project Achilles. By computing the difference in sgRNA quantities between the original pDNA pool and final readcounts from the screen, one can infer the impact of silencing a gene on cell survival. A negative LFC implies the perturbation resulted in cell death, as the number of cells infected with that guide decreased over the course of the screen.
However, this is a rather naive metric as there are other variables to consider, including cutting toxicity (ie. copy number variation) and off target effects. Models like CERES and Chronos attempt to correct for these biases and infer the true gene effect. Here is the Chronos preprint, which may offer some more insight: https://www.biorxiv.org/content/10.1101/2021.02.25.432728v1.full
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Hi,
Thank you so much. That is helpful. Could I further ask what is pDNA described here? is that abbreviation of primary DNA, the DNA of cell lines? and pDNA is not a component of the CRISPR system?
Thank you so much!
Yes, pDNA stands for primary DNA. For each screen we sequence the pDNA pool for the Avana library as a baseline reference for the original sgRNA abundances. Cell lines expressing CRISPR-Cas9 are then transduced with the pDNA pool. The supplementary information in this paper describes the experimental setup for both Achilles and Project Score in more detail: Agreement between two large pan-cancer CRISPR-Cas9 gene dependency data sets | Nature Communications
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Got you! Thank you so much!
