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Gene-based CRISPR score = average guide-based CRISPR score across all sgRNAs targeting a given geneįor each gene, the sgRNA with the lowest overall CRISPR score can be defined as the “best” sgRNA for that gene. Guide-based CRISPR score (overall) = average guide-based CRISPR score across all samples Guide-based CRISPR score (per sample) = log2 Normalize all final samples and the aggregate reference set by reads per million (RPM). One may want to remove control and/or non-genic guides before further analysis. For example, this was set to 200 for AML and 400 for CML.Īdd a pseudocount of 1 to each sgRNA for each sample. This “adequate number” depends on the number of samples and sample coverage. Remove all sgRNAs that don't have an adequate number of counts in the (initial) aggregate reference set.
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Once counts are obtained, replicate measurements were averaged.Īn aggregate reference set (summing initial counts from several samples) may be warranted, based on the experimental design.
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See /nfs/BaRC_Public/BaRC_code/Perl/read_count_CRISPR_guides/read_count_CRISPR_ for one implementation of this. Guide sequences should be required to have an exact match at the expected position (typically at the beginning of the read). Recommendations for this method come from Whitehead Functional Genomics platform.īasic method is described in Wang et al., 2015, Identification and characterization of essential genes in the human genome, Science, 2015 Nov 27, 350(6264).ĭetailed methods are in the supplementary materials. Pooled CRISPR screen analysis Method 1 (based on Wang et al., 2015)