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. 2016 Feb;54(2):78-85.
doi: 10.1002/dvg.22915. Epub 2016 Jan 25.

Chromosome engineering in zygotes with CRISPR/Cas9

Katharina Boroviak  1 , Brendan Doe  1 , Ruby Banerjee  1 , Fengtang Yang  1 , Allan Bradley  1
Affiliations

Chromosome engineering in zygotes with CRISPR/Cas9

Katharina Boroviak et al. Genesis. 2016 Feb.

Abstract

Deletions, duplications, and inversions of large genomic regions covering several genes are an important class of disease causing variants in humans. Modeling these structural variants in mice requires multistep processes in ES cells, which has limited their availability. Mutant mice containing small insertions, deletions, and single nucleotide polymorphisms can be reliably generated using CRISPR/Cas9 directly in mouse zygotes. Large structural variants can be generated using CRISPR/Cas9 in ES cells, but it has not been possible to generate these directly in zygotes. We now demonstrate the direct generation of deletions, duplications and inversions of up to one million base pairs by zygote injection.

Keywords: CRISPR/Cas9; large structural variants; zygote injection.

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Figures

Figure 1
Figure 1
Generation of small deletions using CRISPR/Cas9. (a) Schematic representation of the CRISPR/Cas9 gRNA sites (red arrowheads) for the 9.5 and 65 kb deletions as well as genotyping primer sites (black arrowheads) and oligonucleotides (blue). (b) Deletion junction sequences for the 3 founders for the 9.5 kb deletion and the 13 founders for the 65 kb deletion with gRNA sites indicated in bold and PAM sites in red. The expected gRNA cut site is indicated by red arrowheads. Sequences marked in blue show deletions with defined breakpoints directed by the oligonucleotide.
Figure 2
Figure 2
Generation of large structural variants. (a) Schematic representation of the possible outcomes of genomic rearrangements. gRNA sites are indicated with red arrowheads, genotyping primer sites with black arrowheads. (b) PCR genotyping of DNA from 20 founder mice for the 1.15 Mb genomic rearrangements. Panels 1 and 2, analysis of the breakpoints using primer sets which flank the gRNA cut sites at the proximal and distal ends of the region. Panel 3, deletion breakpoint analysis with FW1 + RV2 primers. Panels 4 and 5, inversion breakpoint analysis at the proximal (FW1 + FW1) and distal (RV1 + RV2) ends of the inversion, respectively.
See this image and copyright information in PMC

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