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Creating cancer translocations in human cells using Cas9 DSBs and nCas9 paired nicks

Benjamin Renouf1, Marion Piganeau1, Hind Ghezraoui1

  • 1Museum National d'Histoire Naturelle, INSERM U1154, CNRS 7196, Paris, France.

Methods in Enzymology
|November 16, 2014
PubMed

Insights

This study introduces methods to create cancer translocations in human cells using Cas9 technology. These techniques enable precise analysis of translocation formation and off-target effects, advancing cancer research.

Area of Science:

  • Molecular biology
  • Genetics
  • Cancer research

Background:

  • Recurrent chromosomal translocations are hallmarks of many cancers, often creating oncogenic fusion genes.
  • Studying translocations at their native sites offers deeper insights than ectopic gene expression.

Purpose of the Study:

  • To present methods for generating cancer-associated chromosomal translocations in human cells.
  • To detail techniques for quantifying translocation frequency and analyzing breakpoint junctions.
  • To demonstrate sensitive detection of off-target effects using PCR.

Main Methods:

  • Induction of translocations using Cas9-generated double-strand breaks (DSBs) or nickase-generated paired nicks at endogenous loci.
  • Quantitative analysis of translocation frequency and breakpoint junction characterization via PCR-based methods.
  • Utilizing PCR for sensitive detection of off-target translocation events.

Main Results:

  • Successful induction of chromosomal translocations at specific genomic locations in human cells.
  • Quantification of translocation frequencies and detailed analysis of resulting breakpoint junctions.
  • Demonstration of PCR's high sensitivity for detecting unintended, off-target translocations.

Conclusions:

  • Cas9-based genome editing provides a powerful platform for creating and studying cancer translocations in relevant cellular contexts.
  • The described methods facilitate the molecular characterization of translocation formation and the assessment of genome editing specificity.
  • This approach enhances the understanding of tumorigenesis driven by chromosomal rearrangements and aids in evaluating gene-editing safety.
Keywords:
Cas9Cas9D10AChromosomal translocationsDNA double-strand breaksEWS–FLI1NPM–ALKPaired nicksnCas9

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