Tandem Repeat Modification during Double-Strand Break Repair Induced by an Engineered TAL Effector Nuclease in Zebrafish Genome

<div><p>Tandem repeats (TRs) are abundant and widely distributed in eukaryotic genomes. TRs are thought to have various functions in gene transcription, DNA methylation, nucleosome position and chromatin organization. Variation of repeat units in the genome is observed in association with a number of diseases, such as Fragile X Syndrome, Huntington's disease and Friedreich's ataxia. However, the underlying mechanisms involved are poorly understood, largely owing to the technical limitations in modification of TRs at definite sites in the genome <i>in vivo</i>. Transcription activator-like effector nucleases (TALENs) are widely used in recent years in gene targeting for their specific binding to target sequences when engineered <i>in vitro</i>. Here, we show that the repair of a double-strand break (DSB) induced by TALENs adjacent to a TR can produce serial types of mutations in the TR region. Sequencing analysis revealed that there are three types of mutations induced by the DSB repair, including indels only within the TR region or within the flanking TALEN target region or simutaneously within both regions. Therefore, desired TR mutant types can be conveniently obtained by using engineered TALENs. These results demonstrate that TALENs can serve as a convenient tool for modifying TRs in the genome in studying the functions of TRs.</p></div

Type of clones in the examined DSTR TALEN individuals.

<p>Type of clones in the examined DSTR TALEN individuals.</p

A (TG)_n sequence in the upstream region of <i>ntl</i> and TALEN targets design.

<p>(A) Position of a (TG)_n sequence in the upstream region of zebrafish <i>ntl</i> and two target sites for designing TALENs. The arrow indicates the transcription start site (TSS) of <i>ntl</i>. The (TG)_n region is showed in grey adjacent boxes, and the two designed TALEN targets are described below, in which red letters underlined represent the binding sites of left (L) and right (R) TALENs, respectively. All the TALEN target sites were designed with a preceding T at 5' terminal (showed in lowercase). (B) Alignment of (TG)_n sequence at upstream region of <i>ntl</i> among zebrafish, bisexual diploid and unisexual polyploid goldfish. GF: bisexual diploid goldfish (<i>Carassius auratus</i>), PZ: unisexual polyploid goldfish (<i>Carassius auratus</i>, pengze), ZF: zebrafish. (C) Structure of TALEN fusion protein, which is composed of a N-terminal translocation domain (NT), a central DNA binding domain, and a C-terminal domain containing a nuclear location site (NLS) and followed by a <i>Fok</i> I nuclease. The TALEN DNA binding domain typically comprises a tandem array of 13–28 single repeat unit <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084176#pone.0084176-Bogdanove1" target="_blank">[22]</a>, each one consisting of 34 highly conserved residues, in which the residues at positions 12 and 13 are called repeat-variable di-residue (RVD). Different RVDs associate specifically with different nucleotides, with NI, NG, HD, and NN accounting for each of the four nucleotides A, T, C and G, respectively. The end of C-terminal repeat unit (showed in the short green box) generally contains only 20 amino acids and is therefore referred to as ‘half-repeat’, which includes a RVD specifically recognizing the nucleotide T.</p

Speculative mechanisms involved in TALEN induced DSB repair.

<p>Blue lines represent the genome with DSB sites, and clusters of vertical bars indicate the TR region. The DSB ends can be bound by two groups of proteins independently: the binding of DNA-dependent protein kinase (DNA-PK) complex (Ku 70 and Ku80) and the following ligase IV seals the gap by direct rejoin the broken ends, which is termed non-homologous end-joining (NHEJ) pathway (A); While the binding of MRN complex and Exo1 nuclease initiates the 5'-3' resection of the ends, which is followed by either a replication slippage pathway (B) or homologous recombination (HR) pathway (C, D). In the replication slippage pathway, the TR region forms a secondary structure and leads to mispairing between the template and the newly-synthesized DNA strand. In the HR pathway, the 3' overhang invades into the homologous template DNA (red lines) and primes DNA synthesis (dash lines) to form a structure called D-loop, which will result in a double Holliday junction (dHJ). dHJ can either be resolved by strand cleavage with or without crossover, which is referred as classical DSB repair (DSBR) pathway of HR (C), and dHJ can also be dissolved by helicases to generate a non-crossover (D). Alternatively, D-loop can be directly dissociated through a synthesis-dependent strand annealing (SDSA) pathway, which results in exclusively non-crossover products (D).</p

Statistics of zebrafish embryos after one day of TALENs injection at different dosage.

<p>The number of embryos scored (N) is indicated at the top and the dosage is indicated at the bottom.</p

Sequencing and Characterization of the Invasive Sycamore Lace Bug <i>Corythucha ciliata</i> (Hemiptera: Tingidae) Transcriptome

<div><p>The sycamore lace bug, <i>Corythucha ciliata</i> (Hemiptera: Tingidae), is an invasive forestry pest rapidly expanding in many countries. This pest poses a considerable threat to the urban forestry ecosystem, especially to <i>Platanus</i> spp. However, its molecular biology and biochemistry are poorly understood. This study reports the first <i>C</i>. <i>ciliata</i> transcriptome, encompassing three different life stages (Nymphs, adults female (AF) and adults male (AM)). In total, 26.53 GB of clean data and 60,879 unigenes were obtained from three RNA-seq libraries. These unigenes were annotated and classified by Nr (NCBI non-redundant protein sequences), Nt (NCBI non-redundant nucleotide sequences), Pfam (Protein family), KOG/COG (Clusters of Orthologous Groups of proteins), Swiss-Prot (A manually annotated and reviewed protein sequence database), and KO (KEGG Ortholog database). After all pairwise comparisons between these three different samples, a large number of differentially expressed genes were revealed. The dramatic differences in global gene expression profiles were found between distinct life stages (nymphs and AF, nymphs and AM) and sex difference (AF and AM), with some of the significantly differentially expressed genes (DEGs) being related to metamorphosis, digestion, immune and sex difference. The different express of unigenes were validated through quantitative Real-Time PCR (qRT-PCR) for 16 randomly selected unigenes. In addition, 17,462 potential simple sequence repeat molecular markers were identified in these transcriptome resources. These comprehensive <i>C</i>. <i>ciliata</i> transcriptomic information can be utilized to promote the development of environmentally friendly methodologies to disrupt the processes of metamorphosis, digestion, immune and sex differences.</p></div

MicroRNA-20a is essential for normal embryogenesis by targeting <i>vsx1</i> mRNA in fish

<div><p></p><p>MicroRNAs are major post-transcriptional regulators of gene expression and have essential roles in diverse developmental processes. In vertebrates, some regulatory genes play different roles at different developmental stages. These genes are initially transcribed in a wide embryonic region but restricted within distinct cell types at subsequent stages during development. Therefore, post-transcriptional regulation is therefore required for the transition from one developmental stage to the next and establishment of different cell identities. However, the regulation of many multiple functional genes at post-transcription level during development remains unknown. Here we show that miR-20a can target the mRNA of <i>vsx1</i>, a multiple functional gene, at the 3′-UTR and inhibit protein expression in both goldfish and zebrafish. The expression of miR-20a is initiated ubiquitously at late gastrula stage and exhibits a tissue-specific pattern in the developing retina. Inhibition of <i>vsx1</i> 3′-UTR mediated protein expression occurs when and where miR-20a is expressed. Decoying miR-20a resulted in severely impaired head, eye and trunk formation in association with excessive generation of <i>vsx1</i> marked neurons in the spinal cord and defects of somites in the mesoderm region. These results demonstrate that miR-20a is essential for normal embryogenesis by restricting Vsx1 expression in goldfish and zebrafish, and explain how transcription factor Vsx1 regulates different development processes.</p></div

Selection and evaluation of reference genes for expression analysis using quantitative real-time PCR in the Asian Ladybird <i>Harmonia axyridis</i> (Coleoptera: Coccinellidae)

<div><p><i>Harmonia axyridis</i> (Coleoptera: Coccinellidae) is a polyphagous insect that is an important biological agent used to control agricultural and forestry pests. The role of functional genes in <i>H</i>. <i>axyridis</i> based on quantitative real-time PCR (qRT-PCR) is increasingly well understood to investigate biology, physiology, feeding behavior and the role of important genes in physiological processes. Quantitative real-time PCR (qRT-PCR) is a powerful and reliable technique to quantify gene expression. Using qRT-PCR, expression levels of target genes are determined based on the levels of internal reference genes; therefore, reference genes need to be stably expressed under specific experimental conditions. However, there have been no studies on the stability of reference genes used in <i>H</i>. <i>axyridis</i>. In this study, we systematically investigated expression profiles of nine candidate reference genes from <i>H</i>. <i>axyridis</i>, including β-actin (<i>ACTIN</i>); elongation factor 1 α (<i>EF1A</i>); ribosomal proteins L10, L18, L28, S13, and S15 (<i>RPL10</i>, <i>RPL18</i>, <i>RPL28</i>, <i>RPS13</i> and <i>RPS15</i>); glyceraldehyde-3-phosphate dehydrogenase (<i>GAPDH</i>); and superoxide dismutase (<i>SOD</i>). Four analytical methods (geNorm, BestKeeper, NormFinder, and the ΔCt method) were used to evaluate the suitability of these genes as internal reference genes for three biotic factors (developmental stage, tissue, and sex) and two abiotic treatments (temperature and photoperiod). RefFinder, a comprehensive evaluation platform integrating the four analytical methods, was used to rank the overall stability of these reference genes. Among the nine candidate genes, different reference genes were identified as having the most stable expression across biotic and abiotic factors. Genes encoding ribosomal proteins typically had the most stable expression, though <i>EF1A</i> was the most stable across developmental stages and photoperiods. To validate the suitability of these reference genes, heat shock protein 90 (<i>HSP90</i>) was chosen as a target. Significant up-regulation in <i>HSP90</i> expression level in response to both low and high temperature was observed when using the most suitable reference genes but not when using an arbitrarily selected reference gene. The reference genes identified in this study will provide the basis for future functional genomics research in <i>H</i>. <i>axyridis</i> and will also facilitate the establishment of a standardized qRT-PCR program for other related insects.</p></div

Stability of reference gene expression across biotic conditions.

<p>Stability of reference gene expression across biotic conditions.</p

Expression profiles of the nine housekeeping genes and the target gene <i>HSP90</i> in <i>H</i>. <i>axyridis</i> under different factors.

<p>Expression level is indicated by cycle threshold (Ct) value. Samples included different development stages, sexes, tissues, photoperiods, and temperatures. Values are means±SD.</p