Optimisation of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 : single-guide RNA (sgRNA) delivery system in a goat model

Huang, Yu; Ding, Yige; Liu, Yao; Zhou, Shiwei; Ding, Qiang; Yan, Hailong; Ma, Baohua; Zhao, Xiaoe; Wang, Xiaolong; Chen, Yulin

doi:10.1071/rd18485

Cited by 4 publications

(5 citation statements)

References 23 publications

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“…Editing efficiency of four sgRNAs (sgRNA 1–4 , sgRNA 1–5 , sgRNA 2–1 , and sgRNA 2–2 ) was greater than 50% (Fig. 1 b), which is consistent with recent studies [ 19 , 39 , 40 ]. Indeed, the in vitro screening of selected sgRNAs is critical for accurately determining the highly efficient sgRNAs required for downstream experiments at the embryonic and animal levels.…”

Section: Resultssupporting

confidence: 92%

“…In this study, sgRNAs with NGG as protospacer adjacent motif (PAM) sequence targeting the sheep MSTN gene (NCBI gene ID: 443449) were designed using sgRNAcas9 and Cas-OFFinder software packages [ 37 – 39 ]. Ten sgRNAs — five sgRNAs located in the first exon, namely, sgRNA 1–1 , sgRNA 1–2 , sgRNA 1–3 , sgRNA 1–4 , and sgRNA 1–5 ; three sgRNAs located in the second exon, namely, sgRNA 2–1 , sgRNA 2–2 , and sgRNA 2–3 ; and two sgRNAs located in the third exon, namely, sgRNA 3–1 and sgRNA 3–2 — were selected which exhibited predicted high-targeting activity and low off-target efficiency (see Additional file 1 : Table S5).…”

Section: Methodsmentioning

confidence: 99%

“…Collected embryos were cytoplasmically coinjected with a mixture of 100 ng/μL of Cas9 mRNA and 200 ng/μL of sgRNAs (as revealed by the optimal result of Group #8 at the embryonic level) using the Eppendorf FemtoJet system. The following parameters were used: microinjection pressure, 45 kPa; compensatory pressure, 7 kPa; and time, 0.1 s. Microinjection was performed in an Olympus ON3 micromanipulation system [ 39 ]. Microinjected embryos were cultured in Quinn’s Advantage Cleavage Medium (Sage) for 24 h and subsequently transferred into surrogates as previously reported [ 19 ].…”

Section: Methodsmentioning

confidence: 99%

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Optimized Cas9:sgRNA delivery efficiently generates biallelic MSTN knockout sheep without affecting meat quality

et al. 2022

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Background CRISPR/Cas9-based genome-editing systems have been used to efficiently engineer livestock species with precise genetic alterations intended for biomedical and agricultural applications. Previously, we have successfully generated gene-edited sheep and goats via one-cell-stage embryonic microinjection of a Cas9 mRNA and single-guide RNAs (sgRNAs) mixture. However, most gene-edited animals produced using this approach were heterozygotes. Additionally, non-homozygous gene-editing outcomes may not fully generate the desired phenotype in an efficient manner. Results We report the optimization of a Cas9 mRNA-sgRNA delivery system to efficiently generate homozygous myostatin (MSTN) knockout sheep for improved growth and meat production. Firstly, an sgRNA selection software (sgRNAcas9) was used to preliminarily screen for highly efficient sgRNAs. Ten sgRNAs targeting the MSTN gene were selected and validated in vitro using sheep fibroblast cells. Four out of ten sgRNAs (two in exon 1 and two in exon 2) showed a targeting efficiency > 50%. To determine the optimal CRISPR/Cas9 microinjection concentration, four levels of Cas9 mRNA and three levels of sgRNAs in mixtures were injected into sheep embryos. Microinjection of 100 ng/μL Cas9 mRNA and 200 ng/μL sgRNAs resulted in the most improved targeting efficiency. Additionally, using both the highly efficient sgRNAs and the optimal microinjection concentration, MSTN-knockout sheep were generated with approximately 50% targeting efficiency, reaching a homozygous knockout efficiency of 25%. Growth rate and meat quality of MSTN-edited lambs were also investigated. MSTN-knockout lambs exhibited increased body weight and average daily gain. Moreover, pH, drip loss, intramuscular fat, crude protein, and shear force of gluteal muscles of MSTN-knockout lambs did not show changes compared to the wild-type lambs. Conclusions This study highlights the importance of in vitro evaluation for the optimization of sgRNAs and microinjection dosage of gene editing reagents. This approach enabled efficient engineering of homozygous knockout sheep. Additionally, this study confirms that MSTN-knockout lambs does not negatively impact meat quality, thus supporting the adoption of gene editing as tool to improve productivity of farm animals.

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Section: Resultssupporting

confidence: 92%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Optimized Cas9:sgRNA delivery efficiently generates biallelic MSTN knockout sheep without affecting meat quality

et al. 2022

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“…Two sgRNAs that showed higher geneediting efficiency in goat parthenogenetic embryos were mixed and used at 25 ng/μL, together with 50 ng/μL Cas9 mRNA for goat zygote injection on the basis of a previous study. 20 Some of the goat zygotes were cultured in G-1 PLUS medium (10128, Vitrolife, Gothenburg, Sweden) for dividing and following in the G-2 PLUS (10132, Vitrolife) medium for developing into blastocysts at 37 °C with 5% CO 2 and others were used for embryo transfer immediately after microinjection. When the cultured embryos developed to blastocysts, the single embryo was used for genomic DNA extraction.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

Knockout of Stearoyl-CoA Desaturase 1 Decreased Milk Fat and Unsaturated Fatty Acid Contents of the Goat Model Generated by CRISPR/Cas9

Tian

Niu

Luo

et al. 2022

J. Agric. Food Chem.

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Goat milk contains a rich source of nutrients, especially unsaturated fatty acids. However, the regulatory mechanism of milk fat and fatty acid synthesis remains unclear. Stearoyl-CoA desaturase 1 (SCD1) is the key enzyme catalyzing monounsaturated fatty acid synthesis and is essential for milk lipid metabolism. To explore milk lipid synthesis mechanism in vivo, SCD1-knockout goats were generated through CRISPR/Cas9 technology for the first time. SCD1 deficiency did not influence goat growth or serum biochemistry. Plasma phosphatidylcholines increased by lipidomics after SCD1 knockout in goats. Whole-blood RNA-seq indicated alterations in biosynthesis of unsaturated fatty acid synthesis, cAMP, ATPase activity, and Wnt signaling pathways. In SCD1-knockout goats, milk fat percentage and unsaturated fatty acid levels were reduced but other milk components were unchanged. Milk lipidomics revealed decreased triacylglycerols and diacylglycerols levels, and the differential abundance of lipids were enriched in glycerolipid, glycerophospholipids, and thermogenesis metabolism pathways. In milk fat globules, the expression levels of genes related to fatty acid and TAG synthesis including SREBP1 were reduced. ATP content and AMPK activity were promoted, and p-p70S6K protein level was suppressed in SCD1-knockout goat mammary epithelial cells, suggesting that SCD1 affected milk lipid metabolism by influencing AMPK-mTORC1/p70S6K-SREBP1 pathway. The integrative analysis of gene expression levels and lipidomics of milk revealed a crucial role of SCD1 in glycerolipids and glycerophospholipids metabolism pathways. Our observations indicated that SCD1 regulated the synthesis of milk fat and unsaturated fatty acid in goat by affecting lipid metabolism gene expression and lipid metabolic pathways. These findings would be essential for improving goat milk nutritional value which is beneficial to human health.

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“…Ten healthy ewes (3 to 5 years old) with normal estrous cycles were selected as donors for zygote collection. The superovulation treatment of the donors was performed as previously described [40]. Briefly, an EAZI-BREED controlled internal drug release (CIDR) Sheep and Goat Device (containing 300 mg of progesterone) was inserted into the vagina of the donor ewes for 12 days and superovulation was performed 60 h prior to the removal of the CIDR Device.…”

Section: Production Of Mutated Sheepmentioning

confidence: 99%

Highly efficient generation of sheep with a defined FecBB mutation via adenine base editing

et al. 2020

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Base editing has the potential to improve important economic traits in agriculture and can precisely convert single nucleotides in DNA or RNA sequences into minimal double-strand DNA breaks (DSB). Adenine base editors (ABE) have recently emerged as a base editing tool for the conversion of targeted A:T to G:C, but have not yet been used in sheep. ABEmax is one of the latest versions of ABE, which consists of a catalytically-impaired nuclease and a laboratory-evolved DNA-adenosine deaminase. The Booroola fecundity (FecB B) mutation (g.A746G, p.Q249R) in the bone morphogenetic protein receptor 1B (BMPR1B) gene influences fecundity in many sheep breeds. In this study, by using ABEmax we successfully obtained lambs with defined point mutations that result in an amino acid substitution (p.Gln249Arg). The efficiency of the defined point mutations was 75% in newborn lambs, since six lambs were heterozygous at the FecB B mutation site (g.A746G, p.Q249R), and two lambs were wild-type. We did not detect off-target mutations in the eight edited lambs. Here, we report the validation of the first gene-edited sheep generated by ABE and highlight its potential to improve economically important traits in livestock.

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Optimisation of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 : single-guide RNA (sgRNA) delivery system in a goat model

Cited by 4 publications

References 23 publications

Optimized Cas9:sgRNA delivery efficiently generates biallelic MSTN knockout sheep without affecting meat quality

Optimized Cas9:sgRNA delivery efficiently generates biallelic MSTN knockout sheep without affecting meat quality

Knockout of Stearoyl-CoA Desaturase 1 Decreased Milk Fat and Unsaturated Fatty Acid Contents of the Goat Model Generated by CRISPR/Cas9

Highly efficient generation of sheep with a defined FecBB mutation via adenine base editing

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