Dipak Pandey, Takahiro Matsubara, Taiju Saito, Yukinori Kazeto, Koichiro Gen, Tetsushi Sakuma, Takashi Yamamoto, Miyuki Mekuchi, Rie Goto

J. Mar. Sci. Eng. 2021, 9 (12), 1378

Published: 4 December 2021; https://doi.org/10.3390/jmse9121378

Abstract

Transcription activator-like effector (TALE) nucleases (TALENs) mediated gene editing methods are becoming popular and have revealed the staggering complexity of genome control during development. Here, we present a simple and efficient gene knockout using TALENs in kawakawa, Euthynnus affinis, using slc24a5. We examined slc24a5 gene expression and functional differences between two TALENs that hold the TALE scaffolds, +153/+47 and +136/+63 and target slc24a5. Developmental changes in slc24a5 transcripts were seen in early-stage embryos by real-time PCR; slc24a5 expression was first detected 48 h post fertilization (hpf), which increased dramatically at 72 hpf. Four TALENs, 47- and 63-type of two different target loci (A and B), respectively, were constructed using Platinum TALEN and evaluated in vitro by a single-strand annealing (SSA) assay. TALEN activities were further evaluated in vivo by injecting TALEN mRNAs in the two-cell stage of the zygote. Most of the TALEN-induced mutants showed mosaic patterns in the retinal pigment epithelium (RPE) and fewer melanin pigments on the body at 72 hpf and later when compared to the control, implying the gene’s association with melanin pigment formation. A heteroduplex mobility assay (HMA) and the genome sequence further confirmed the TALEN-induced mutations of substitution, insertion, and deletion at an endogenous locus.

See: https://www.mdpi.com/2077-1312/9/12/1378

Figure 1. Design of slc24a5 TALENs in kawakawa, Euthynnus affinis. (a) Schematic representation of the structure of the complete sequence of the slc24a5 gene. Coding and untranslated exons regions are shown as solid and open boxes, respectively. The number of nucleotides present in each exon and intron is shown. (b) slc24a5 TALENs designed to target exon1 of the gene. The exon1 sequence was determined based on the genomes of five different individuals. The grey shaded and underlined sequences indicate the TALEN binding sites of targets A and B, respectively.