title: Enriching CRISPR-Cas9 targeted cells by co-targeting the HPRT gene.
abstract: The CRISPR-Cas9 system uses guide RNAs to direct the Cas9 endonuclease to cleave target sequences. It can, in theory, target essentially any sequence in a genome, but the efficiency of the predicted guide RNAs varies dramatically. If no targeted cells are obtained, it is also difficult to know why the experiment fails. We have developed a transient transfection based method to enrich successfully targeted cells by co-targeting the hypoxanthine phosphoribosyltransferase (HPRT) gene. Cells are transfected with two guide RNAs that target respectively HPRT and the gene of interest. HPRT targeted cells are selected by resistance to 6-thioguanine (6-TG) and then examined for potential alterations to the gene targeted by the co-transfected guide RNA. Alterations of many genes, such as AAVS1, Exo1 and Trex1, are highly enriched in the 6-TG resistant cells. This method works in both HCT116 cells and U2OS cells and can easily be scaled up to process multiple guide RNAs. When co-targeting fails, it is straightforward to determine whether the target gene is essential or the guide RNA is ineffective. HPRT co-targeting thus provides a simple, efficient and scalable way to enrich gene targeting events and to identify the cause of failure.
authors.affiliation: Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA.
authors.forename: Shuren
authors.initials: S
authors.lastname: Liao
authors.affiliation: Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA.
authors.forename: Margaret
authors.initials: M
authors.lastname: Tammaro
authors.affiliation: Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA Hong.Yan@fccc.edu.
authors.forename: Hong
authors.initials: H
authors.lastname: Yan
mesh_terms: D064113:CRISPR-Cas Systems; D045744:Cell Line, Tumor; D005091:Exons; D018390:Gene Targeting / Q000379:methods*; D020043:Genes, Essential; D056426:Genetic Loci; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D012313:RNA / Q000378:metabolism; D014162:Transfection
chemical_list: D012313:RNA; D007041:Hypoxanthine Phosphoribosyltransferase