Comparison of gene editing efficiencies of CRISPR/Cas9 and TALEN for generation of MSTN knock-out cashmere goats
Introduction
Alpas cashmere goats, which adapt well to the semi-arid temperate grassland, are the most precious genetic resources in China. High yield and good quality is one of the vital aims of cashmere goat breeding. Myostatin (MSTN), also known as growth and differentiation factor 8, is a member of the transforming growth factor β (TGF-β) superfamily. It is a negative regulator of skeletal muscle development and its suppression results in enhanced muscle growth and increased leanness of carcass composition [1]. MSTN inactivation is the molecular cause of the double muscling phenotype in the Belgian Blue and Piedmontese breeds of beef cattle [2]. Recent studies showed that inhibiting MSTN increases skeletal muscle mass, reduces fat mass, and inhibits diet-induced and genetic obesity [3], providing an opportunity to increase muscle growth and improve meat production by genetic manipulation in livestock [4].
Relying on natural mutations for selective animal breeding is most often impractical because they occur randomly, at low frequencies, and require long-term phenotype screening. The use of conventional hybridization breeding for introducing preexisting mutations is also time-consuming, especially in large, genetically complex domestic animals. In recent years, genome editing using site-specific nucleases, such as zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) [5] has gained popularity for use in cell lines, animals, and plants [[6], [7], [8]]. These methods enhance the homologous recombination (HR) and non-homologous end joining (NHEJ) efficiency by inducing highly specific DNA double-strand breaks (DSB) at the targeted position of the genome [9,10]. TALEN has low toxicity and is very efficient, very specific and rarely shows off-target effects [11]. In contrast, CRISPR/Cas9, despite being more efficient than TALEN, may generate higher off-target effects [12]. Both methods have their own advantages and disadvantages; the differences in efficiency between CRISPR/Cas9 and TALEN should be tested for each target locus in each target cell type to assess the usefulness of these tools for each objective [13].
In this study, we have compared the efficiencies of gene targeting and editing achieved by TALEN and CRISPR/Cas9 in editing the MSTN gene. Our study aimed to identify the robust and efficient gene-engineering tool that can be applied for breeding of farm animals, and therefore will be critically important and applicable for breeding. We also describe the strategy used for the successful generation of a cloned goat.
Section snippets
Ethics statement
This study was carried out in accordance with the recommendations of the National Research Council Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Institutional Animal Care and Use Committee of Inner Mongolia University (Hohhot, China). All animals were maintained at the Inner Mongolia YiWei White Cashmere Goat Limited Liability Company.
Cell isolation and culture
Primary embryonic fibroblast cells of Alpas cashmere goat were isolated from goat fetuses on day 40. The fetal body was
Optimizing transfection in goat embryonic fibroblasts (GEFs) by CRISPR/Cas9 or TALEN
The low transfection efficiency of primary cells has always been a challenge in large animal transgene research, especially in marker-free clean genetic modification. Several studies have focused on this problem, and many transfection strategies including lipid-based delivery, electroporation and nucleofection, have been applied to goat primary fibroblasts [19]. In this study, fluorescence measurements by flow cytometry showed that the highest electroporation efficiency was 47.7%, the
Discussion
Engineered, site-specific nuclease-induced genomic double-strand DNA breaks and break repair processes enable genome editing in a plethora of eukaryotic genomes [[20], [21], [22]]. Commonly used genome editing systems include ZFN [23], TALEN [24], and CRISPR/Cas9 [20,25]. TALENs and CRISPR/Cas9 are much more efficient than ZFN for genome editing, as they are easier to manipulate and have greatly reduced the cost of genome modification [21,26]. The application of genome editing has become
Acknowledgments
This work was supported by The Science and Technology Innovation Guided Project in Inner Mongolia Autonomous Region, China (KCBJ2018003). We are grateful to the Inner Mongolia YiWei White Cashmere Goat limited liability company for their support with the embryo-transfer experiments.
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