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.
Journal article
The CRISPR journal
12/2019
2
406 - 416
Cancer Genetics and Evolution Laboratory, Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom; University of Oxford, Oxford, United Kingdom.
Cell Line, Tumor, Humans, Neoplasms, Chromosomal Instability, Cytogenetic Analysis, Gene Rearrangement, Mutagenesis, Sequence Deletion, Mutation, CRISPR-Cas Systems, Clustered Regularly Interspaced Short Palindromic Repeats, Gene Editing, RNA, Guide, Kinetoplastida