Time origin and structural analysis of the induced CRISPR/cas9 megabase-sized deletions and duplications involving the Cntn6 gene in mice

Abstract

In a previous study using one-step CRISPR/Cas9 genome editing in mouse zygotes, we created five founders carrying a 1,137 kb deletion and two founders carrying the same deletion, plus a 2,274 kb duplication involving the Cntn6 gene (encoding contactin-6). Using these mice, the present study had the following aims: (i) to establish stage of origin of these rearrangements; (ii) to determine the fate of the deleted DNA fragments; and (iii) to estimate the scale of unpredicted DNA changes accompanying the rearrangements. The present study demonstrated that all targeted deletions and duplications occurred at the one-cell stage and more often in one pronucleus only. FISH analysis revealed that there were no traces of the deleted DNA fragments either within chromosome 6 or on other chromosomes. These data were consistent with the Southern blot analysis showing that chromosomes with deletion often had close to expected sizes of removed DNA fragments. High-throughput DNA sequencing of two homozygotes for duplication demonstrated that there were no unexpected significant or scale DNA changes either at the gRNA and joint sites or other genome sites. Thus, our data suggested that CRISPR/Cas9 technology could generate megabase-sized deletions and duplications in mouse gametes at a reasonably specific level.

Targeted genome editingin vivocorrects aDmdduplication restoring wild‐type dystrophin expression

“…Our initial choice of employing multiple sgRNAs to cleave the duplicated region has been proposed to enhance the likelihood of obtaining the desired rearrangement (Boroviak et al, 2016). Indeed, we observed the presence of duplication junctions in approximately 1% of the screened clones, confirming the intrinsic complexity of generating this structural variant (Kraft et al, 2015;Boroviak et al, 2016;Pristyazhnyuk et al, 2019). However, achieving the desired duplication was accompanied by a second, undesired structural variation, as previously described by others (Shin et al, 2017;Kosicki et al, 2018).…”

“…Duplication disorders, such as MDS, PMD, and CMT, have been primarily investigated using transgenic animals overexpressing the causative genes (Kagawa et al, 1994; Magyar et al, 1996; Collins et al, 2004). In addition, chromosomal engineering via the Cre‐Lox system and, more recently, genome engineering have been used to generate some mouse models involving tandem multi‐gene duplications (Walz et al, 2006; Li et al, 2007; Nakatani et al, 2009; Yu et al, 2010; Horev et al, 2011; Clark et al, 2013; Pristyazhnyuk et al, 2019). With regard to intragenic duplications, the only available model to date is the Dup2 model affecting the Dmd gene (Vulin et al, 2015).…”

On-Target CRISPR/Cas9 Activity Can Cause Undesigned Large Deletion in Mouse Zygotes

“…Nowadays, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system, known as the most powerful tool for gene editing, is intensively used in a wide range of biological studies, for conducting various screenings on cell cultures [1][2][3][4] and creating unique genome-modified animal models [5][6][7][8]. To date, two major strategies have been successfully developed and used to create genome-modified animals by using the CRISPR/Cas9 system.…”

Characterization of Poldip2 knockout mice: Avoiding incorrect gene targeting

“…Another anecdotal piece of evidence regarding the frequent occurrence of long duplications comes from another recent project, carried out for our laboratory by another company, in which a line of knockin mice also failed to produce Hom pups and had to be derived from another ES cell clone. It should be noted that megabase duplications are also observed after repair of double strand breaks induced by CRISPR/Cas9 [ 61 , 62 ].…”