Deletions, Inversions, Duplications: Engineering of Structural Variants using CRISPR/Cas in Mice

Abstract

Structural variations (SVs) contribute to the variability of our genome and are often associated with disease. Their study in model systems was hampered until now by labor-intensive genetic targeting procedures and multiple mouse crossing steps. Here we present the use of CRISPR/Cas for the fast (10 weeks) and efficient generation of SVs in mice. We specifically produced deletions, inversions, and also duplications at six different genomic loci ranging from 1.1 kb to 1.6 Mb with efficiencies up to 42%. After PCR-based selection, clones were successfully used to create mice via aggregation. To test the practicability of the method, we reproduced a human 500 kb disease-associated deletion and were able to recapitulate the human phenotype in mice. Furthermore, we evaluated the regulatory potential of a large genomic interval by deleting a 1.5 Mb fragment. The method presented permits rapid in vivo modeling of genomic rearrangements.

Genome editing for the reproduction and remedy of human diseases in mice

“…Another method to generate large duplications may be introduction of Cas9/a pair of sgRNAs into ES cells instead of fertilized eggs, since the number of clones to be screened can be easily increased when ES cells are used [35]. A disadvantage of this approach is that it takes a longer time for the generation of mice with mutations from ES cells compared to that of zygote injection.…”

Correction of the exon 2 duplication in DMD myoblasts by a single CRISPR/Cas9 system

“…As previously reported by Kraft et al, 34 the use of two synthetic gRNAs targeting two different positions of the same chromosome induces deletions, inversions and duplications through non-homologous end joining (NHEJ). Hence, we tested the occurrence of deletions and inversions by specifically designed PCR ( Figures 2C and 2D) amplifying and sequencing the expected deletions and inversions of different range between the target sites of the tested gRNAs ( Figures 2E and 2F).…”

“…Interestingly, we did not detect inversions when dup2 patient's myoblasts were treated, backing up the concept that deletions occur more frequently than inversions in our system, as described elsewhere. 34 Treatment of immortalized myoblasts derived from two dup2 patients resulted in the deletion of the duplication at the genomic level ( Figure 3A) and in an increased expression of wild-type dystrophin transcripts ( Figure 3B). This expression was followed by a detectable and correctly localized dystrophin only in the #994 cell line ( Figures 3C and 3D), which carries a smaller duplication of 137 kb, and not in the #515 cell line, which carries a larger, 263-kb duplication, probably due to the limited transduction efficiency in this cell line.…”

CRISPR/Cas9-Facilitated Chromosome Engineering to Model Human Chromosomal Alterations

“…Current data have not provided suicient evidence to draw such a conclusion [7][8][9]. On the other hand, current data do suggest that chromosomal location and/or the endpoints of the fragment manipulated through CRISPR/Cas9 can inluence the eiciency of genome engineering.…”

“…Other engineered mouse mutants, which carry a triplication or deiciency of smaller Hsa21 syntenic regions [5,6], have facilitated systematic genetic dissections of DS phenotypes. With the emergence of CRISPR/Cas9-facilitated genome editing, atempts have been made to further improve the eiciency of mammalian chromosome manipulations, whether it be deletions, duplications, inversions, or translocations [7][8][9][10], including those in Hsa21 syntenic regions.…”