Elsevier

Theriogenology

Volume 132, 1 July 2019, Pages 1-11
Theriogenology

Comparison of gene editing efficiencies of CRISPR/Cas9 and TALEN for generation of MSTN knock-out cashmere goats

https://doi.org/10.1016/j.theriogenology.2019.03.029Get rights and content

Highlights

  • We successfully generated an MSTN KO lamb using CRISPR/Cas9.

  • We compared utility of CRISPR/Cas9 and TALEN for generating Alpas cashmere goats with myostatin (MSTN) gene knockout (KO).

  • Compared with CRISPR/Cas9 embryos, TALEN MSTN KO embryos reached 8-cell stage at significantly higher cleavage rate.

  • Compared with TALEN, CRISPR/Cas9 showed greater gene editing efficiency, but with higher number of off-target effects.

  • CRISPR/Cas9 has high potential to become a robust gene-engineering tool for application farm animal breeding.

Abstract

The genome editors CRISPR/Cas9 (clustered regularly interspaced short palindromicrepeats/Cas9 nuclease-null) and TALENs (transcription activator-like effector nuclease) are popularly used for targeted modification of the mammalian genome. To date, few comparative studies have been carried out to investigate the differences between the use of CRISPR/Cas9 and TALENs in genome editing for goat breeding. Here, we compared CRISPR/Cas9 and TALEN technologies at multiple levels for generating a knock out (KO) of the Alpas cashmere goat myostatin (MSTN) gene, which negatively regulates the proliferation and differentiation of skeletal muscle cells. The electrotransfection efficiency observed using CRISPR/Cas9 was 8.1% more than that observed using TALEN for generating MSTN KO cells. In addition, the cutting efficiency of CRISPR/Cas9 for editing exon 1 of the MSTN gene was higher than that of TALENs. However, the off-target effects of the CRISPR/Cas9 system were also higher than those of TALENs. Further, we found that the frequency of obtaining MSTN−/- mutations by CRISPR/Cas9 was 8.5 times higher than that by TALEN. The CRISPR/Cas9-edited colonies involved longer deletions (up to 117 bp) than the TALEN-edited colonies (up to 13 bp). Remarkably, when embryos used to generate cloned goat via somatic cell nuclear transfer were compared, we found that the TALEN MSTN KO embryos easily developed to 8 cells and their cleavage rate was significantly higher than that of CRISPR/Cas9-edited embryos. Finally, we produced a MSTN KO lamb using CRISPR/Cas9, which suggested that a high level of targeted gene modification could be achieved in goat using CRISPR/Cas9. Taken together, our study indicates that although TALEN enables a variety of genome modifications and may have some advantages over CRISPR/Cas9, the latter provides a significant advantage by permitting precise and efficient gene editing. Thus, CRISPR/Cas9 has more potential to become a robust gene-engineering tool for application in the breeding of farm animals.

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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