Deletion of miR-142-3p in miR-142a and miR-142b double mutant zebrafish. (A) Target sites in zebrafish miR-142a and amino acid sequences of the zinc-finger proteins were selected to recognize this site by OPEN. Spacer sequences in the target site are shown in lowercase letters. Each of the selected zinc-finger arrays targeting 9-bp nucleotides was fused with a subunit of the FokI nuclease. (B) The target site in zebrafish miR-142b and the amino acid sequences of the zinc-finger proteins were designed to recognize this site by CoDA. (C) Secondary structure prediction of the pre-miR-142a in the wild-type 142a^wt (left) or 142 a^m1 (right) allele. Mature wild-type miR-142-3p from pre-miR-142a is boxed with a red dashed line. (D) Secondary structure prediction of pre-miR-142b in wild-type miR-142b^wt (left) or miR-142b^m1 (right) allele. Mature wild-type miR-142-3p from pre-miR-142b is boxed with a red dashed line. (E) The expression level of miR-142-3p in wild-type, 142 a^m1/m1 (142a^−/−), and 142 a^m1/m1142b^m1/m1 (142ab^−/−) mutant zebrafish embryos at 3 dpf, which was detected by real-time PCR. The expression of miR-142-3p and miR-142a-5p is normalized to glyceraldehyde-3-phosphate dehydrogenase (n = 3 for each genotype). Error bars indicate the standard error of the mean (SEM). **P < .01 compared with wild-type control. (F-H) Detection of miR-142-3p expression in wild-type, 142a^−/−, and 142ab^−/− mutant zebrafish embryos at 5 dpf by WISH. The black triangle indicates the thymus; yellow triangle, kidney; and red triangle, CHT. Note that the staining was weakened, but not diminished, in the 142a^−/− zebrafish embryos. (I-J) In vivo reporter assays for the interaction between miR-142-3p and the targets with wild-type pre-miR-142a/b^wt or mutant pre-miR-142a/b^m1. EGFP mRNA harboring 2 perfect complementary target sites (EGFP-2 x PT) was coinjected with pre-miR-142a^wt (I), pre-miR-142 a^m1 (I), pre-miR-142b^wt (J), or pre-miR-142b^m1(J); DsRed mRNA served as a control.