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Enriching CRISPR-Cas9 targeted cells by co-targeting the HPRT gene
Shuren Liao1, Margaret Tammaro1, Hong Yan2
1Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA.
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.
Insights
CRISPR-Cas9 gene editing efficiency is improved by co-targeting the hypoxanthine phosphoribosyltransferase (HPRT) gene. This method enriches successfully edited cells and helps identify ineffective guide RNAs or essential target genes.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- CRISPR-Cas9 gene editing relies on guide RNAs to direct Cas9 cleavage.
- Guide RNA efficiency varies, making it hard to troubleshoot failed experiments.
- Identifying reasons for CRISPR-Cas9 failure, such as ineffective guide RNAs or essential target genes, is challenging.
Purpose of the Study:
- To develop a method for enriching cells successfully targeted by CRISPR-Cas9.
- To provide a way to distinguish between ineffective guide RNAs and essential target genes.
- To improve the efficiency and scalability of CRISPR-Cas9 gene editing experiments.
Main Methods:
- Developed a transient transfection method co-targeting the hypoxanthine phosphoribosyltransferase (HPRT) gene alongside the gene of interest.
- Utilized two guide RNAs: one targeting HPRT and another targeting the gene of interest.
- Selected HPRT-targeted cells by resistance to 6-thioguanine (6-TG) for analysis of gene of interest alterations.
Main Results:
- Successfully enriched gene-targeted cells in both HCT116 and U2OS cell lines.
- Demonstrated enrichment of alterations in genes like AAVS1, Exo1, and Trex1 in 6-TG resistant cells.
- Established a scalable method applicable to multiple guide RNAs and genes.
Conclusions:
- Co-targeting HPRT provides a robust strategy to enrich CRISPR-Cas9 gene editing events.
- This method simplifies troubleshooting by differentiating between essential target genes and ineffective guide RNAs.
- Offers a simple, efficient, and scalable approach to enhance gene targeting success.
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