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Nucleic Acids Res. 2015 Nov 16;43(20):e134. doi: 10.1093/nar/gkv675. Epub 2015 Jun 29.

Enriching CRISPR-Cas9 targeted cells by co-targeting the HPRT gene.

Author information

1
Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA.
2
Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA Hong.Yan@fccc.edu.

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.

PMID:
26130722
PMCID:
PMC4787791
DOI:
10.1093/nar/gkv675
[Indexed for MEDLINE]
Free PMC Article

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