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

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.

Cas9-induced large deletions and small indels are controlled in a convergent fashion

“…These effects were also noted in conjunction with templated editing in mice (33)(34)(35)(36). Extensive loss-of-heterozygosity by gene conversion and megabase-long deletions were also observed (37)(38)(39)(40). Such outcomes could be pathogenic, and may be hard to genotype.…”

Unintended on-target chromosomal instability following CRISPR/Cas9 single gene targeting

“…Two studies 5 , 6 previously reported incidental chromosome arm truncation following CRISPR/Cas9 targeting telomere-proximal genes: POLE 5 located 11 kb and UROS 6 located 6 million bp away from chromosome arm end. ZNF516 is located 6 million bp from the telomere, whereas MEX3C , targeting which did not induce arm truncation, is located 31 million bp away from the arm end.…”

“…Reports by Rayner et al. 5 and Cullot et al. 6 suggested detection of arm truncation with FISH analysis for targeted genes; however, this can only be achieved after cell colony expansion and involves several experimental steps.…”

KLF17 promotes human naïve pluripotency but is not required for its establishment

“…For the remaining 4 clones, the damage apparently completely prevented amplification of the second allele. This highlights the importance of in-depth genotyping following CRISPR-Cas9-mediated mutagenesis, as previously noted (Kosicki et al, 2018, Cullot et al, 2019, Rayner et al, 2019, Przewrocka et al, 2020. We therefore sought to test whether clones #18 and #19, compound mutants with two frameshifted alleles predicted to introduce premature stop codons in the sequence encoding the third zinc fingers (FIG.S7D-F), were null for KLF17 expression.…”