مهمترین تکنیک‌های ویرایش ژن و ژنوم

نوع مقاله : مقاله پژوهشی

نویسندگان

1 گروه بیوتکنولوژی کشاورزی، دانشکده کشاورزی، دانشگاه تربیت مدرس، تهران، ایران

2 گروه بیوتکنولوژی کشاورزی، دانشگده کشاورزی، دانشگاه تربیت مدرس، تهران، ایران

3 مختار جلالی جواران، گروه بیوتکنولوژی کشاورزی، دانشکده کشاورزی، دانشگاه تربیت‌مدرس، تهران، ایران

چکیده

هدف: این مقاله مروری کلی بر تاریخچه کشف ، مکانیسم عملکرد، مزایا، محدودیت ها و چالش‌ها، کاربرد‌ها و چشم‌اندازهای آینده این فناوری ها ارائه می دهد تا اطلاعاتی مفید و خلاصه شده را در اختیار پژوهشگران قرار دهد.
مواد و روش ها: ویرایش هدفمند ژنوم مبتنی بر ایجاد شکست دو‌ رشته‌ای و به دنبال آن ترمیم رشته مورد‌نظر از طریق مکانیسم‌های ترمیمی نوترکیبی همولوگ (HDR) و یا اتصال انتها‌های غیر‌همولوگ (NHEJ) است که در بسیاری از موجودات زنده جهت اهداف مختلف مورداستفاده قرار گرفته است. به‌ منظور انجام ویرایش ژنی، اولین نوکلئاز اختصاصی MegaN بود که در سال 1985 کشف شد و پس از آن با کشف موتیف ZF، نوکلئاز‌ ZFN و TALEN و CRISPR و Fanzor توسعه یافتند که برای ویرایش ژنوم موجودات زنده مورد استفاده قرار گرفتند. این نوکلئاز‌ها با قرار‌گیری روی رشته DNA و شناسایی توالی هدف و اتصال به آن‌ها، با استفاده از دُمین نوکلئازی برش را ایجاد می‌کنند. با کشف سیستم CRISPR/Cas دوره جدیدی از تحقیقات در زمینه ویرایش ژن آغاز شد. مکانیسم عمل در این روش، شناسایی توالی هدف توسط این نوکلئاز بر اساس برهم‌کنش DNA و رشته RNA راهنما است که پس از شناسایی توالی هدف، برش توسط دُمین نوکلئازی انجام می‌شود.
نتایج: از مثال‌های موفقیت‌آمیز در استفاده از این تکنیک‌ها می‌توان به درمان برخی از بیماری‌ها اشاره کرد: در سال 2018 بیماری سندروم هانتر با وارد کردن ژن IDS به سلول‌های کبد با استفاده از روش ZFN به‌صورت درون‌تنی درمان شد. از روش TALEN برای درمان بیماری دیستروفی عضلانی دوشن استفاده شد. در سال 2021 دانشمندان توانستند با تکنیک ویرایش تک باز، بیماری کم خونی داسی شکل را با بهره‌گیری از سیستم CRISPR/Cas درمان کنند.
بحث: در روش‌های نوین ویرایش ژن و ژنومی، پروتئین‌های نوکلئاز‌ی قادرند به طور هدفمند تغییراتی مانند درج، حذف و یا جایگزینی در توالی نوکلئوتیدی، ویرایش اطلاعات ژنتیکی و اپی‌ژنوم را انجام دهند. این ابزار‌ها می توانند از طریق ویرایش ژن‌های عامل بیماری‌زا از طریق خاموش کردن آن‌ها و یا فعال‌کردن ژن‌های مهارکننده عامل بیماری‌ در حوزه‌های کشاورزی، پزشکی و درمان بیماری‌های ژنتیکی به کار برده شوند. چنین سیستم هایی، در نشانه‌گذاری و همچنین کاهش یا افزایش بیان ژن‌ها کاربرد مؤثری دارند. با توسعه روش‌های جدید‌تر و دقیق‌تر، استفاده از نوکلئاز‌های MegaN کاهش یافت و بیشتر مطالعات و آزمایش‌ها بر روی روش‌های TALEN و CRISPR/Cas متمرکز گردیده است.

کلیدواژه‌ها

عنوان مقاله [English]

The most important techniques for gene and genome editing

نویسندگان [English]

  • Mokhtar Jalali Javaran 1
  • Maryam Mohaghegh 2
  • Sedighe Setayesh 1
  • Mahsa Zarei 3
  • Sina Nosrat abady 1
1 Department of Agricultural Biotechnology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran
2 Department of Agricultural Biotechnology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran
3 Department of Agricultural Biotechnology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran
چکیده [English]

Objecrive
This article aims to provide a comprehensive overview of the history of the discovery of these tools, their mechanisms of action, along with their advantages, disadvantages, challenges, applications, and future prospects, presented in a concise and informative manner for enthusiasts.
Materials and methods
Targeted genome editing is based on the induction of double-strand breaks, followed by the repair of the desired strand through mechanisms such as homologous recombination repair (HDR) or non-homologous end joining (NHEJ), which have been utilized across various organisms for diverse purposes. The first specialized nuclease, MegaN, was discovered in 1985, marking the inception of gene editing. Subsequently, the discovery of the ZF motif led to the development of ZFN, TALEN, CRISPR, and Fanzor nucleases, which have been employed for genome editing in living organisms. These nucleases function by binding to the DNA strand, identifying the target sequence, and, through their nuclease domain, introducing a cut. The discovery of the CRISPR/Cas system heralded a new era in gene editing research. The mechanism of action in this system involves the identification of the target sequence by the nuclease through the interaction of DNA and a guide RNA strand; following target sequence identification, the cut is made by the nuclease domain.
Results
Successful examples of these techniques include the treatment of certain diseases: in 2018, Hunter syndrome was treated by introducing the IDS gene into liver cells using the ZFN method in vivo. The TALEN method was used to treat Duchenne muscular dystrophy. In 2021, scientists were able to treat sickle cell anemia using the CRISPR/Cas system through single-base editing techniques.
Conclusions
In modern gene and genome editing methodologies, nuclease proteins are capable of introducing targeted modifications such as insertions, deletions, or substitutions within nucleotide sequences, thereby editing genetic information and the epigenome. These tools are capable of editing pathogenic genes by silencing them or activating inhibitory genes, finding applications in agriculture, medicine, and the treatment of genetic diseases. This system is also effective in gene tagging and modulating gene expression levels. With the development of newer and more precise methods, the use of MegaN nucleases has declined, and research has increasingly focused on TALEN and CRISPR/Cas methods.

کلیدواژه‌ها [English]

  • CRISPR/Cas
  • gene and genomic editing
  • TALEN
  • ZFN

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مجله بیوتکنولوژی کشاورزی
دوره 18، شماره 2
خرداد 1405
صفحه 41-68

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