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Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9
Published on: January 3, 2015
Engineering large-scale chromosomal deletions by CRISPR-Cas9
Thomas F Eleveld1, Chaimaa Bakali1, Paul P Eijk1
1Department of Pathology, Cancer CenterAmsterdam, Amsterdam UMC, Vrije Universiteit Amsterdam, De Boelelaan 1117, 1081 HV Amsterdam, the Netherlands.
Abstract:
Large-scale chromosomal deletions are a prevalent and defining feature of cancer. A high degree of tumor-type and subtype specific recurrencies suggest a selective oncogenic advantage. However, due to their large size it has been difficult to pinpoint the oncogenic drivers that confer this advantage. Suitable functional genomics approaches to study the oncogenic driving capacity of large-scale deletions are limited. Here, we present an effective technique to engineer large-scale deletions by CRISPR-Cas9 and create isogenic cell line models. We simultaneously induce double-strand breaks (DSBs) at two ends of a chromosomal arm and select the cells that have lost the intermittent region. Using this technique, we induced large-scale deletions on chromosome 11q (65 Mb) and chromosome 6q (53 Mb) in neuroblastoma cell lines. A high frequency of successful deletions (up to 30% of selected clones) and increased colony forming capacity in the 11q deleted lines suggest an oncogenic advantage of these deletions. Such isogenic models enable further research on the role of large-scale deletions in tumor development and growth, and their possible therapeutic potential.
Insights
Researchers engineered large-scale chromosomal deletions using CRISPR-Cas9 technology. This method effectively created cancer cell models, revealing deletions that confer an oncogenic advantage in tumor development.
Area of Science:
- Oncology
- Genomics
- Molecular Biology
Background:
- Large-scale chromosomal deletions are common in cancer, suggesting they provide a selective oncogenic advantage.
- Identifying specific oncogenic drivers within these large deletions is challenging due to their size.
- Limited functional genomics tools exist for studying the impact of large deletions.
Purpose of the Study:
- To develop an effective CRISPR-Cas9 based technique for engineering large-scale chromosomal deletions.
- To create isogenic cell line models harboring these deletions for functional studies.
- To investigate the oncogenic role and therapeutic potential of large deletions.
Main Methods:
- Simultaneous induction of double-strand breaks (DSBs) at two chromosomal locations using CRISPR-Cas9.
- Selection of cells with the intervening chromosomal region deleted.
- Application of the technique to engineer 65 Mb deletions on chromosome 11q and 53 Mb deletions on chromosome 6q in neuroblastoma cell lines.
Main Results:
- Successful engineering of large-scale deletions with a high frequency (up to 30% of selected clones).
- Neuroblastoma cell lines with 11q deletions exhibited increased colony-forming capacity, suggesting an oncogenic advantage.
- The developed method effectively generated isogenic cell line models for studying large deletions.
Conclusions:
- The CRISPR-Cas9 technique provides an effective means to engineer large chromosomal deletions and create valuable isogenic models.
- Large deletions, such as those on chromosome 11q, can confer an oncogenic advantage in cancer.
- These isogenic models are crucial for further research into the role of large deletions in tumorigenesis and their therapeutic implications.
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