Genome-Wide CRISPR/Cas9 Screening for Identification of Cancer Genes in Cell Lines.
Methods in molecular biology (Clifton, N.J.)(2019)
摘要
In this protocol, pooled sgRNA libraries targeting thousands of genes are computationally designed, generated using microarray-based synthesis techniques, and packaged into lentiviral particles. Target cells of interest are transduced with the lentiviral sgRNA pools to generate a collection of knockout mutants-via Cas9-mediated genomic cleavage-and screened for a phenotype of interest. The relative abundance of each mutant in the population can be monitored over time through high-throughput sequencing of the integrated sgRNA expression cassettes. Using this technique, we outline strategies for the identification of cancer driver genes and genes mediating drug response.
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关键词
CRISPR/Cas9 mutagenesis screens,Drug sensitivity,Loss-of-function gene discovery,sgRNA libraries
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