Targeted disruption of sp7 and myostatin with CRISPR-Cas9 results in severe bone defects and more muscular cells in common carp.

Zhaomin Zhong,Shuhao Xu,Yilin Yan,Han Wang,Yi Hou,Yi Sun,Xiaowen Sun,Pengfei Niu,Mingyong Wang,Guodong Huang,Xiaona Xu

doi:10.1038/srep22953

Abstract

The common carp (Cyprinus carpio) as one of the most important aquaculture fishes produces over 3 million metric tones annually, approximately 10% the annual production of the all farmed freshwater fish worldwide. However, the tetraploidy genome and long generation-time of the common carp have made its breeding and genetic studies extremely difficult. Here, TALEN and CRISPR-Cas9, two versatile genome-editing tools, are employed to target common carp bone-related genes sp7, runx2, bmp2a, spp1, opg, and muscle suppressor gene mstn. TALEN were shown to induce mutations in the target coding sites of sp7, runx2, spp1 and mstn. With CRISPR-Cas9, the two common carp sp7 genes, sp7a and sp7b, were mutated individually, all resulting in severe bone defects; while mstnba mutated fish have grown significantly more muscle cells. We also employed CRISPR-Cas9 to generate double mutant fish of sp7a;mstnba with high efficiencies in a single step. These results demonstrate that both TALEN and CRISPR-Cas9 are highly efficient tools for modifying the common carp genome, and open avenues for facilitating common carp genetic studies and breeding.

Highlights

The common carp (Cyprinus carpio) as one of the most important aquaculture fishes produces over 3 million metric tones annually, approximately 10% the annual production of the all farmed freshwater fish worldwide
It appears that CRISPR-Cas[9] has advantages of ease to carry out, high efficiencies and low cost over TALEN13,15,25,28–31, and CRISPR-Cas[9] allows for mutagenizing multiple genes in the stem cells or zygotes, and generating bialleic mutants with clear phenotypes in the F0 generation for studying gene functions without crossing animals for several generations[24,32,33,34]. Both methods have not been applied in common carp to date. We have employed both TALEN and CRISPR-Cas[9] techniques to modify genes involved in bone formation such as sp[7], a zinc-finger-containing transcription factor expressed in osteoblasts[35], which can activate pre-osteoblast cells to differentiate into mature osteoblast cells and osteocytes[36,37]
We generated double mutants of mstnba;sp7a in common carp with high efficiencies. These results demonstrate that both TALEN and CRISPR-Cas[9] systems are effective genome-editing tools for common carp genetic studies and breeding

Summary

Introduction

The common carp (Cyprinus carpio) as one of the most important aquaculture fishes produces over 3 million metric tones annually, approximately 10% the annual production of the all farmed freshwater fish worldwide. Evaluate mutagenesis efficiencies of TALEN and CRISPR-Cas[9] of common carp genes using zebrafish embryos. Because of the ease of zebrafish embryos to work with, we first used them to evaluate mutagenesis efficiencies of TALENs and CRISPR-Cas9/gRNAs of common carp (Fig. 2A).

Results

Conclusion