CRISPR-Cas9 Causes Chromosomal Instability and Rearrangements in Cancer Cell Lines, Detectable by Cytogenetic Methods

CRISPR J. 2019 Dec;2(6):406-416. doi: 10.1089/crispr.2019.0006. Epub 2019 Nov 19.

Abstract

CRISPR-Cas9 has quickly become the method of choice for genome editing, with multiple publications describing technical advances and novel applications. It has been widely adopted as a tool for basic research and has significant translational and clinical potential. However, its usage has outpaced the establishment of essential and rigorous controls for unwanted off-target effects, manifested as small mutations, large deletions of target loci, or large-scale chromosomal rearrangements. A common application of CRISPR-Cas9 is as a tool for creating isogenic cell-line models to study the effects of precise mutations, or variants, on disease traits. Here, we describe the effect of standard CRISPR-Cas9 mutagenesis protocols on well characterized cancer cell lines. We demonstrate that commonly used methods for detecting correctly mutated clones fail to uncover large-scale rearrangements. We show that simple cytogenetic methods can be used to identify clones carrying chromosomal abnormalities and large mutations at target loci. These methods are quick and cost-efficient, and we suggest that such controls should be performed prior to publication of studies based on novel CRISPR-Cas9 mutated cancer cell lines.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems / genetics
  • Cell Line, Tumor
  • Chromosomal Instability / genetics*
  • Chromosomal Instability / physiology
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics
  • Cytogenetic Analysis / methods*
  • Gene Editing / methods*
  • Gene Rearrangement / genetics
  • Humans
  • Mutagenesis / genetics
  • Mutation
  • Neoplasms / genetics
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • Sequence Deletion / genetics

Substances

  • RNA, Guide, CRISPR-Cas Systems