Heritable Targeted Inactivation of Myostatin Gene in Yellow Catfish (Pelteobagrus fulvidraco) Using Engineered Zinc Finger Nucleases

Yellow catfish (Pelteobagrus fulvidraco) is one of the most important freshwater aquaculture species in China. However, its small size and lower meat yield limit its edible value. Myostatin (MSTN) is a negative regulator of mammalian muscle growth. But, the function of Mstn in fish remains elusive. To explore roles of mstn gene in fish growth and create a strain of yellow catfish with high amount of muscle mass, we performed targeted disruption of mstn in yellow catfish using engineered zinc-finger nucleases (ZFNs). Employing zebrafish embryos as a screening system to identify ZFN activity, we obtained one pair of ZFNs that can edit mstn in yellow catfish genome. Using the ZFNs, we successfully obtained two founders (Founder July29-7 and Founder July29-8) carrying mutated mstn gene in their germ cells. The mutated mstn allele inherited from Founder July29-7 was a null allele (mstnnju6) containing a 4 bp insertion, predicted to encode function null Mstn. The mutated mstn inherited from Founder July29-8 was a complex type of mutation (mstnnju7), predicted to encode a protein lacking two amino acids in the N-terminal secretory signal of Mstn. Totally, we obtained 6 mstnnju6/+ and 14 mstnnju7/+ yellow catfish. To our best knowledge, this is the first endogenous gene knockout in aquaculture fish. Our result will help in understanding the roles of mstn gene in fish

Efficient manipulation of gene expression using Natronobacterium gregoryi Argonaute in zebrafish

Abstract Background Natronobacterium gregoryi Argonaute (NgAgo) was found to reduce mRNA without generating detectable DNA double-strand breaks in a couple of endogenous genes in zebrafish, suggesting its potential as a tool for gene knockdown. However, little is known about how it interacts with nucleic acid molecules to interfere with gene expression. Results In this study, we first confirmed that coinjection of NgAgo and gDNA downregulated target genes, generated gene-specific phenotypes and verified some factors (including 5’ phosphorylation, GC ratio, and target positions) of gDNAs affecting gene downregulation. Therein, the sense and antisense gDNAs were equally effective, suggesting that NgAgo possibly binds to DNA. NgAgo-VP64 with gDNAs targeting promoters upregulated the target genes, further providing evidence that NgAgo interacts with genomic DNA and controls gene transcription. Finally, we explain the downregulation of NgAgo/gDNA target genes by interference with the process of gene transcription, which differs from that of morpholino oligonucleotides. Conclusions The present study provides conclusions that NgAgo may target genomic DNA and that target positions and the gDNA GC ratio influence its regulation efficiency

Summary of the identification of the yellow catfish carrying disrupted <i>mstn</i>.

<p>Summary of the identification of the yellow catfish carrying disrupted <i>mstn</i>.</p

Zebrafish embryos can be used as an <i>in vivo</i> system to examine ZFN activity of editing yellow catfish <i>mstn</i> gene.

<p>(A) Schematic diagram shows ZFN1 binding to the yellow catfish <i>mstn</i> gene. Yellow catfish <i>mstn</i> exons are shown as boxes and its introns are shown as solid lines. Start codon (ATG) and stop codon (TGA) are marked in exon 1 and exon 3, respectively. Number above the exon box denotes the position of nucleotides in the gene <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028897#pone.0028897-Pan1" target="_blank">[4]</a>. The ZFN1 binding site is in exon 1. The triplets of nucleotides recognized by ZFN1 fingers are marked in different colors. (B) Zebrafish embryos were used as an <i>in vivo</i> system to examine ZFN activity of editing yellow catfish <i>mstn</i> gene. The plasmid containing exon 1 of yellow catfish <i>mstn</i> gene (pGEM-ycMSTN) was co-microinjected with ZFN1 mRNA into zebrafish embryos at 1–2-cell stage. The <i>mstn</i> molecules were amplified from the zebrafish embryos at 24 hpf and then subcloned for sequencing. Analyses on sequences of the molecules revealed that the molecules of disrupted <i>mstn</i> were categorized into three groups including deletions, insertions and complex. (C) ZFN1 cut <i>mstn</i> in yellow catfish genome. ZFN1 mRNA was microinjected into yellow catfish embryos at 1–2-cell stage. The <i>mstn</i> molecules were amplified from the yellow catfish embryos at 72 hpf and then subcloned for sequencing. Analyses on sequences of the molecules revealed that the molecules of disrupted <i>mstn</i> in yellow catfish genome were categorized into three groups including deletions, insertion and complex. WT: partial sequence of wild type <i>mstn</i> containing ZFN1 targeting site (B, C). Number in the leftmost of the panels (B, C) shows the number of nucleotides was deleted (−) or inserted (+) in the mutated <i>mstn</i> gene. Number in the bracket shows the frequency of the mutated molecules (B, C). Inserted nucleotides are bolded (B, C).</p

Systematic genome editing of the genes on zebrafish Chromosome 1 by CRISPR/Cas9

Genome editing by the well-established CRISPR/Cas9 technology has greatly facilitated our understanding of many biological processes. However, a complete whole-genome knockout for any species or model organism has rarely been achieved. Here, we performed a systematic knockout of all the genes (1333) on Chromosome 1 in zebrafish, successfully mutated 1029 genes, and generated 1039 germline-transmissible alleles corresponding to 636 genes. Meanwhile, by high-throughput bioinformatics analysis, we found that sequence features play pivotal roles in effective gRNA targeting at specific genes of interest, while the success rate of gene targeting positively correlates with GC content of the target sites. Moreover, we found that nearly one-fourth of all mutants are related to human diseases, and several representative CRISPR/Cas9-generated mutants are described here. Furthermore, we tried to identify the underlying mechanisms leading to distinct phenotypes between genetic mutants and antisense morpholino-mediated knockdown embryos. Altogether, this work has generated the first chromosome-wide collection of zebrafish genetic mutants by the CRISPR/Cas9 technology, which will serve as a valuable resource for the community, and our bioinformatics analysis also provides some useful guidance to design gene-specific gRNAs for successful gene editing