The CRISPR/Cas9 system has emerged as an invaluable tool for generating knockout cell lines. Using an easy-to-source guide RNA moiety combined with an RNA-guided endonuclease to generate targeted DNA damage leading to frameshift mutations is now a facile methodology. However, single cell clone generation and validation is still a time consuming, labor-intensive process. In an effort to adopt a higher throughput, parallelized approach to quickly interrogate whole pathways or cellular processes we looked to repurpose arrayed CRISPR guide RNA screening libraries. Utilizing a combination of automation and next generation sequencing we have developed a method for high-throughput generation and validation of panels of knock-out cell lines. Using this technique it is possible to create and validate a large number of clonal genetic knockouts from a single basal cell background in parallel.
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