Methods and systems for crispr selection

A method comprising:

• summing (Σ) respective numbers of guide RNAs (gRNAs) (n) qualitatively present in a first culture of cas9-positive cells after infection with a library of viral vectors, per target region of DNA, whose read count exceeds a background threshold, wherein the library comprises at least 3 gRNAs for cleaving a target region of DNA within the genome of the cells;
• and
• summing (Σ) a total number of gRNAs (N) qualitatively present, over all target regions, whose read count exceeds the background threshold;
• categorizing, based on applying a selection mechanism to a second culture of cas9-positive cells after infection with the library of viral vectors, the cells of the second culture as either having a designated phenotype or not having the designated phenotype, wherein cas9-positive cells of the second culture having the designated phenotype express a selectable marker comprising a protein of a signaling pathway;
• summing (Σ) respective numbers of gRNAs (n′) qualitatively present, per target region of DNA, in the cas9-positive cells of the second culture having the designated phenotype, whose post-selection read count exceeds the background threshold;
• and
• summing (Σ) a total number of gRNAs (N′) qualitatively present, over all target regions, of the cas9-positive cells of the second culture having the designated phenotype, whose read count exceeds the background threshold;
• calculating, for a target region of DNA, a probability of observing n′ gRNAs for the target region in the cas9-positive cells of the second culture having the designated phenotype by chance according to the formula
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• computes a number of ways of choosing x objects out of y objects;
• calculating, for a target region of DNA that comprises a gene, a probability of observing n′ or more gRNAs of the gene in the cas9-positive cells of the second culture having the designated phenotype by chance according to the formula
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• identifying, based on the probability of observing n′ or more gRNAs of the gene in the cas9-positive cells of the second culture having the designated phenotype by chance, that the gene impacts the signaling pathway.

A method comprising:

• summing respective numbers of gRNAs qualitatively present in cas9-positive cells of a first culture after infection with a library of viral vectors, per target region of DNA, whose read count exceeds a background threshold, wherein the library comprises at least 3 gRNAs for cleaving a target region of DNA within the genome of the cas9-positive cells;
• and
• summing a total number of gRNAs qualitatively present in cas9-positive cells of the first culture after infection with the library of viral vectors, over all target regions, whose read count exceeds the background threshold;
• categorizing, based on applying a selection mechanism to a second culture of cas9-positive cells after infection with the library of viral vectors, the cas9-positive cells of the second culture as either having a designated phenotype or not having the designated phenotype, wherein cas9-positive cells of the second culture having the designated phenotype express a selectable marker comprising a protein of a signaling pathway;
• summing respective numbers of gRNAs qualitatively present, per target region of DNA, in the cas9-positive cells of the second culture having the designated phenotype, whose post-selection read count exceeds the background threshold;
• and
• summing a total number of gRNAs qualitatively present, over all target regions, in the cas9-positive cells of the second culture having the designated phenotype, whose read count exceeds the background threshold;
• calculating, for a target region of DNA, a probability of observing a number of gRNAs qualitatively present for the target region in the cas9-positive cells of the second culture having the designated phenotype, by chance;
• calculating, for a target region of DNA that comprises a gene, a probability of observing a number of gRNAs of the gene in the cas9-positive cells of the second culture having the designated phenotype by chance;
• and
• identifying, based on the probability of observing the number gRNAs of the gene in the cas9-positive cells of the second culture having the designated phenotype by chance, that the gene impacts the signaling pathway.