Abstract
The use of TALEN and CRISPR/CAS nucleases is becoming increasingly popular as a means to edit single target sites in one-cell mouse embryos. Nevertheless, an area that has received less attention concerns the engineering of structural genome variants and the necessary religation of two distant double-strand breaks. Herein, we applied pairs of TALEN or sgRNAs and Cas9 to create deletions in the Rab38 gene. We found that the deletion of 3.2 or 9.3kb, but not of 30kb, occurs at a frequency of 6–37%. This is sufficient for the direct production of mutants by embryo microinjection. Therefore, deletions up to ∼10kb can be readily achieved for modeling human disease alleles. This work represents an important step towards the establishment of new protocols that support the ligation of remote DSB ends to achieve even larger rearrangements.
Highlights
Since 1988, gene targeting in embryonic stem (ES) cells has enabled the generation of mouse mutants and the study of gene function by reverse genetics [1]
Sequence-specific nucleases such as TAL effector nucleases (TALEN) and the CRISPR/Cas system are increasingly used for editing of single target sites in the mouse genome for the introduction of small deletions or point mutations, e.g. for modeling human disease mutations
We found that a 10 kb deletion could be generated in 6% of pups derived from microinjections of two TALEN pairs and that two sgRNAs enabled the deletion of a 3.2 kb gene segment in 37% of mice, but a 30 kb deletion was not obtained among 38 mice
Summary
Since 1988, gene targeting in embryonic stem (ES) cells has enabled the generation of mouse mutants and the study of gene function by reverse genetics [1]. It has transpired that sequence-specific nucleases can be used as an alternative to induce targeted double-strand breaks (DSB) and enhance local DNA repair. It is possible to produce mutants from one cell embryos, independent of ES cells [2]. ⇑ Corresponding authors at: Institute of Developmental Genetics, Helmholtz. Max-Delbrück-Centrum für Molekulare Medizin, Robert-Roessle-Str. 10, 13125 Berlin, Germany.
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