Chromosome engineering in zygotes with CRISPR/Cas9

Abstract

SUMMARYDeletions, 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. genesis 54:78–85, 2016. © 2016 The Authors. genesis Published by Wiley Periodicals, Inc.

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

“…Over the course of this experiment, we also obtained mice carrying a 5 Mb inversion but no duplications were obtained [32]. Boroviak et al [33] also reported that they microinjected Cas9/two sgRNAs and ssODN as a donor to guide deletion in fertilized eggs and detected 1.1 Mb deletions and inversions in 30 and 21% of embryos genotyped, respectively, although no duplications were observed. When they tried to generate 155 and 545 kb rearrangements using the same strategy, only 1-2% of mice analyzed had the duplication, while 18-24 and 18-30% of mice contained deletions and inversions, respectively.…”

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

“…Speciically, it may ofer opportunities to obtain mutants for other less commonly used animal models by direct zygote injection of the CRISPR/Cas9 components without involving ES cells [12][13][14]. This new approach may also facilitate chromosome engineering in mice from diferent strain backgrounds than those currently available with the Cre/loxP-mediated ES cell methods [7,8]. Furthermore, it may lead to the generation of desired animal models more quickly.…”

“…Furthermore, it may lead to the generation of desired animal models more quickly. Table 1 summarized the eiciencies of three types of structural variations, deletion, inversion, and duplication, in mouse [7,8].…”

“…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.…”

Combinations of chromosome transfer and genome editing for the development of cell/animal models of human disease and humanized animal models

“…Now, the combination of chromosome transfer and genome editing technology is crucial for the efficient production of fully humanized animal models (Fig. 4) [76]. If genome editing of KO endogenous genes can be applied to germlinetransmitted Tc animals, both time and cost for mating Tc and KO animals will be saved.…”