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Updated: Mar 30, 2026
Pooled CRISPR-Based Genetic Screens in Mammalian Cells
Published on: September 4, 2019
A Medium-Throughput Single Cell CRISPR-Cas9 Assay to Assess Gene Essentiality
A R Grassian1, T M E Scales2, S K Knutson1
1Epizyme, Inc., 400 Technology Square, 4th Floor, Cambridge, MA 02139 USA.
Background:
Target selection for oncology is a crucial step in the successful development of therapeutics. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 editing of specific loci offers an alternative method to RNA interference and small molecule inhibitors for determining whether a cell line is dependent on a specific gene product for proliferation or survival. In our initial studies using CRISPR-Cas9 to verify the dependence on EZH2 activity for proliferation of a SMARCB1/SNF5/INI1 mutant malignant rhabdoid tumor (MRT) cell line, we noted that the initial reduction in proliferation was lost over time. We hypothesized that in the few cells that retain proliferative capacity, at least one allele of EZH2 remains functional. To verify this, we developed an assay to analyze 10s-100s of clonal cell populations for target gene disruption using restriction digest and fluorescent fragment length analyses.
Results:
Our results clearly show that in cell lines in which EZH2 is essential for proliferation, at least one potentially functional allele of EZH2 is retained in the clones that survive.
Conclusion:
This assay clearly indicates whether or not a specific gene is essential for survival and/or proliferation in a given cell line. Such data can aid the development of more robust therapeutics by increasing confidence in target selection.
Insights
CRISPR-Cas9 gene editing helps identify essential genes for cancer cell survival. This study developed a new assay to confirm if a gene, like EZH2, is critical for proliferation, aiding therapeutic development.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Target selection is critical for oncology therapeutic development.
- CRISPR-Cas9 gene editing is an alternative to RNA interference and small molecule inhibitors for functional gene analysis.
- Initial CRISPR-Cas9 studies showed transient proliferation reduction in EZH2-dependent malignant rhabdoid tumor (MRT) cell lines.
Purpose of the Study:
- To develop and validate a novel assay for analyzing gene disruption in clonal cell populations.
- To confirm the hypothesis that surviving cells retain at least one functional EZH2 allele.
- To determine gene essentiality for cell line proliferation and survival.
Main Methods:
- Utilized Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 gene editing.
- Developed a clonal cell population assay.
- Employed restriction digest and fluorescent fragment length analyses to detect target gene disruption.
Main Results:
- The developed assay confirmed that EZH2 is essential for proliferation in specific cell lines.
- In EZH2-dependent cell lines, surviving clones retained at least one functional EZH2 allele.
- The assay effectively identified gene essentiality for cell proliferation.
Conclusions:
- The assay reliably indicates whether a gene is essential for cell survival and/or proliferation.
- This method enhances confidence in target selection for oncology drug development.
- Robust target validation aids the creation of more effective therapeutics.
