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The CRISPR-Cas9 system uses guide RNA to direct the Cas9 endonuclease to introduce DNA double-strand breaks (DSB) at practically any specified site in the genome, followed by non-homologous end joining or homology-dependent repair systems, leading to the inactivation of the target gene or its editing, respectively. The power of the CRISPR has made it possible to disrupt and modify all of the predicted genes in the human genome. Despite the success of CRISPR, the efficiency still varies greatly due to guide RNA effectiveness, transfection efficiency, retrovirus titer, and cell type. This problem is compounded exponentially when one needs to target sequentially two or more genes (iterative gene targeting) to analyze their functional relationship.
Researchers from Fox Chase Cancer Center have discovered that co-targeting the hypoxanthine phosphoribosyltransferase (HPRT) gene with gene of interest, followed by selection, leads to dramatic enrichment of cells with mutations in the gene of interest. Uniquely, the HPRT gene can be disrupted and corrected in a sequential way by CRISPR-induced non- homologous repair and homology-dependent repair. Both steps can be selected with drugs and thus used for co-targeting of different genes, making it easy to construct mutant cells with as many genes disrupted as cell viability permits. This novel method has several major advantages:
Liao S et al. Enriching CRISPR-Cas9 targeted cells by co-targeting the HPRT gene. Nucleic Acids Res. 2015 Nov 16;43(20):e134.
Patent Status: A patent application has been filed.
For Licensing/Partnering information, please contact:
Inna Khartchenko, MS, MBA
Director, Technology Transfer and New Ventures
215-214-3989
Inna.Khartchenko@fccc.edu