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.
Summary of the Invention
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:
- It is highly efficient, usually approaching 100% in most cell lines tested.
- It only uses basic tissue culture setup and standard transfection reagents. No sophisticated equipment, expensive reagents, or complicated protocols are required.
- It is easy to scale up simply by increasing the number of transfections.
- It does not involve viruses.
- It leaves no genetic “scars” other than changes in the non-essential HPRT gene and the genes of interest.
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.
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Inna Khartchenko, MS, MBA
Director, Technology Transfer and New Ventures