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FEBS Open Bio. 2014 Dec 3;5:26-35. doi: 10.1016/j.fob.2014.11.009. eCollection 2015.

Creation of targeted genomic deletions using TALEN or CRISPR/Cas nuclease pairs in one-cell mouse embryos.

Author information

1
Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, 85764 Neuherberg, Germany ; Technische Universität München, 85350 Freising-Weihenstephan, Germany.
2
Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, 85764 Neuherberg, Germany.
3
Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, 85764 Neuherberg, Germany ; Deutsches Zentrum für Neurodegenerative Erkrankungen e.V. (DZNE), 80336 Munich, Germany.
4
Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, 85764 Neuherberg, Germany ; Technische Universität München, 85350 Freising-Weihenstephan, Germany ; Deutsches Zentrum für Neurodegenerative Erkrankungen e.V. (DZNE), 80336 Munich, Germany ; Max-Planck-Institute of Psychiatry, 80804 Munich, Germany ; Munich Cluster for Systems Neurology (SyNergy), Ludwig-Maximilians-Universität München, 80336 Munich, Germany.
5
Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, 85764 Neuherberg, Germany ; Technische Universität München, 85350 Freising-Weihenstephan, Germany ; Max-Delbrück-Centrum für Molekulare Medizin (MDC), 13125 Berlin, Germany.

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 9.3 kb, but not of 30 kb, occurs at a frequency of 6-37%. This is sufficient for the direct production of mutants by embryo microinjection. Therefore, deletions up to ∼10 kb 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.

KEYWORDS:

CRISPR; Cas9; DSB, double-strand break; Disease model; ES, embryonic stem (cell); HR, homologous recombination; Mouse mutant; NHEJ, non-homologous end joining; One-cell embryo; TAL, transcription activator-like; TALEN, TAL effector nucleases; TALENs; ZFN, zinc-finger nucleases; sg, single guide

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