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.