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ناک‌اوت ژن میوستاتین‎ ‎با استفاده از فناوری ‏CRISPR/Cas9‎‏ به‌منظور تولید رویان گوسفند ورامینی ‏ناک‌اوت‌شده

نوع مقاله : مقاله پژوهشی

نویسندگان

1 گروه علوم دامی، دانشکده علوم دامی و غذایی، دانشگاه علوم کشاورزی و منابع طبیعی خوزستان، ملاثانی، ایران. رایانامه: m‌-_bazgiri_1989@asnrukh.ac.ir

2 نویسنده مسئول، مرکز تحقیقات سلولی و مولکولی دانشگاه علوم پزشکی شهید بهشتی، تهران، ایران. رایانامه: m.salehi@sbmu.ac.ir

3 گروه بیوتکنولوژی پزشکی، دانشکده فناوری‌های نوین پزشکی، دانشگاه علوم پزشکی شهید بهشتی، تهران، ایران. رایانامه: s.jajarmi@sbmu.ac.ir

10.22059/jap.2024.370186.623774

چکیده

ژن میوستاتین (MSTN)) نقش مهمی در تنظیم توده عضلانی اسکلتی ایفا می‌کند و مهار ترجمه‌ای این ژن نشان‌دهنده افزایش توده عضلانی است که به‌عنوان "فنوتیپ دو ماهیچه‌ای" شناخته می‌شود. اختلال در بیان ژن MSTN با استفاده از تکنولوژی ویرایش ژنوم CRISPR/Cas9 رشد عضلانی و نرخ رشد را در گونه‌های دام از جمله گوسفند و بز نشان داده است. این پژوهش به‌منظور تولید رویان‌‌های گوسفند ورامینی حامل ژن MSTN  ناک‌اوت شده و بررسی این رویان‌‌ها انجام شد. در مرحله اول، برای طراحی و انتخاب RNA راهنما از نرم‌افزار CRISPOR (http://crispor.tefor.net/) برای غربالگری اولیه استفاده شد. دو RNA راهنما که اگزون یک ژن MSTN را هدف قرار می‌دهند انتخاب شدند. سپس به میزان 50 نانوگرم از مخلوط دو RNA راهنما به‌همراه Cas9 mRNA به‌صورت هم‌زمان به تخمک‌‌ها ریز تزریق شد. از بین 12 رویان تولیدشده پنج رویان مارکر تکنولوژی CRISPR را نشان دادند. نتایج توالی‌یابی نشان داد دو نوع جهش برای RNA راهنما یک و RNA راهنما دو به‌صورت جداگانه یافت شد، اما هر دو نوع از RNA راهنماها به‌صورت هم زمان در هیچ‌یک از رویان‌ها عملکرد نداشتند. درصد برش سلول با سایت Synthego، 83 درصد گزارش شد. توالی نوکلئوتیدی اگزون یک MSTN ثبت‌شده در NCBI با توالی گوسفند ورامینی متفاوت بود. براساس نتایج حاصل، بعد از جهش ایجاد شده در ژن MSTN با سیستم CRISPR/Cas9  توالی اسیدآمینه‌ای رویان‌های ناک‌اوت شده ژن MSTN در مقایسه با توالی اسد آمینه­ای گروه کنترل کد خاتمه را نشان دادند.

کلیدواژه‌ها

عنوان مقاله [English]

Knockout of myostatin (MSTN) gene using CRISPR/Cas9 technology in order to produce knockout Varamini sheep embryos

نویسندگان [English]

  • maryam bazgiri 1
  • Jamal Fayazi 2
  • mohammad salehi 2
  • vahid jajarmi 3

1 Department of Animal Science, Faculty of Animal and Food Science, Agricultural Sciences and Natural Resources University of Khuzestan, Mollasani, Iran. E-mail: m‌‌_bazgiri_1989@asnrukh.ac.ir

2 Corresponding Author, Department of Animal Science, Faculty of Animal and Food Science, Agricultural Sciences and Natural Resources University of Khuzestan, Mollasani, Iran. E-mail: j_fayazi@asnrukh.ac.ir

3 Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. E-mail: s.jajarmi@sbmu.ac.ir

چکیده [English]

Introduction: Myostatin (MSTN) is a member of transforming growth factor-β (TGF-β), which is a negative regulator for muscle differentiation and growth in various mammals and plays a key role in muscle growth and meat quality. Today, CRISPR technology can be used to accurately change any attribute. The CRISPR/Cas9 technology creates double-strand breaks (DSBs) in the target region of DNA, which can be repaired by homology repair (HDR) in the presence of the corresponding homologous repair template or by non-homologous end joining (NHEJ).
Materials and Methods: Guide RNAs (sgRNA) were designed using CRISPOR online software. Eggs collected from 150 Varami sheep were placed in 50-microliter drops of culture medium in an incubator containing 7% CO2 and 95% humidity. 22-24 hours after IVM, a mixture of two guide RNAs cloned in a CRISPR vector was injected into each oocyte at a concentration of 30 ng with a microinjection microscope. After microinjection, the parthenogenesis method was used to fertilize the eggs. After the activation of the eggs in the wells of the 96-well plate, the bottom of which was covered with cumulus cells and sage medium for eight days in an incubator with a temperature of 38.5 degrees Celsius and 7% CO2gas, they were cultivated under conditions of maximum humidity. After eight days, the zygotes that had reached the embryonic stage were analyzed with a fluorescent microscope. The embryos of the test group that emitted green light, as well as one embryo from the control group, were individually placed in nine microliters of DNA Lysis to prepare their genomes. They were incubated in a temperature program of one hour at 65 degrees and ten minutes at 90 degrees. In order to investigate the gene editing of the embryos that emitted green light, the PCR products of five greened embryos along with one embryo of the control group were sequenced by the trench method.
Results and Discussion: Finally, 12 sheep embryos were produced, which were analyzed with a fluorescent microscope, and a total of five embryos emitted green light. The green light indicated that they had received the CRISPR/Cas9 technology. Among the five embryos, two of the embryos with guide RNA 1 showed a single nucleotide deletion upstream of PAM. Additionally, two of the embryos showed a single nucleotide deletion in guide RNA 2, while one of the embryos remained unchanged. Sequence analysis of the knockout embryos revealed that 83% of the cells were cut. After creating two types of single nucleotide deletions in different positions of sheep embryos, the effect of this genomic editing was detected by examining the amino acid sequence of the embryos in the control group and those carrying the mutation. It was observed that the deletion of a single nucleotide caused by guide RNA resulted in a change in the genome framework and termination code, leading to a shorter amino acid sequence in the edited sheep compared to the control group. This research marked the first time that laboratory embryos of Varamin sheep genetically manipulated by CRISPR/Cas9 technology were produced.
Conclusion: The nucleotide sequence of MSTN gene in Varami sheep was different from the sequence recorded in NCBI. Five embryos showed CRISPR technology markers. Both of the designed guide RNAs caused mutations in the nucleotide sequence and termination code in the amino acid sequence.

کلیدواژه‌ها [English]

  • CRISPR/Cas9
  • Gene editing
  • MSTN
  • Muscle
  • Varamini sheep
مراجع
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دوره 26، شماره 2
تیر 1403
صفحه 99-110
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