The deletion of a large genetic locus is achieved by the expression of two sets of TALENs by oocyte injection.

(A) TALENs were designed to disrupt one of the exons of mars2-like (mars2-l) gene. PCR primers were used to confirm gene knock-out. Exons are marked by boxes. (B) The injection of TALEN mRNAs into GV oocytes (blue bar), but not into fertilized embryos (red bar), showed better knock-out of the mars2-l exon, as revealed by qPCR analyses. Control represents embryos injected only with right TALEN mRNA. N = 3–4 independent experiments. Error bars are standard errors. *P < 0.05. **P < 0.01. (C) Expression of TALENs before fertilization allowed the production of an embryo carrying only the deleted mars2-l exon in the F0 generation (lane 3, 7 and 8 in TALEN B-D, red color). Genomic DNA was extracted from single embryos at St. 10.5–11 and subjected to PCR analysis. M represents 1.5 kp and 100 bp ladder marker. (D) Large deletions of exon 2 in mars-l were confirmed by sequencing. Sample numbers correspond to those in Fig 4C. A part of inverted mars-l sequence was inserted in sample #3.