Name: crispr-cas9 gene editing in action
Uploaded: 2019-03-11T09:21:22.000Z
Duration: 1 min 59 s
Channel: JoVE

K12 Standards

Tennessee Academic Standards

High School

Biology

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Links Among Engineering, Technology, Science, and Society

BIO1.ETS2.3

CRISPR

نظام كريسبر Cas9 هي أداة تحرير الحمض النووي التي تقف ل متفاوت المسافات بانتظام يتكرر متآلف قصير و CRISPR المرتبطة البروتين 9. لاحظ لأول مرة في البكتيريا كريسبر Cas9 هو وسيلة للدفاع ضد الفيروسات. كما يدخل الحمض النووي الفيروسي الأجنبي بكتيريا يتم معالجتها إلى أجزاء أصغر.التي يمكن إدراجها في المنطقة من الجينوم البكتيري المسمى كريسبر موضع. عندما يتم نسخ المنطقة المنتج يرتبط مع الحمض النووي الريبي أصغر تسمى tracrRNAs والتي قد تساعد في توجيه كل من بروتين Cas9 و RNAse إلى الجزيء. الأخير الذي يشق نسخة.النتيجة النهائية هي العديد من المجمعات كل يتكون من بروتين Cas9 tracrRNA و crRNA المستمدة من الحمض النووي في المكان. يعترف كريسبر RNA في هذه الهياكل ويرشد Cas9 إلى الحمض النووي الفيروسي الذي هو ثم المشقوق وتدميرها. يسخر العلماء كريسبر Cas9 عن طريق تجميع جزيئات الحمض النووي الريبي الفردية أن تقليد tracrRNA و CRISPR RNA والتي يمكن أن تستهدف الجينات ذات الاهتمام.على سبيل المثال عندما اثنين من هذا دليل الحمض النووي الريبي يتم إدخالها في الخلايا مع Cas9 وكلاهما يستهدف نفس الجين تسلسل يمكن رفعه. بمجرد إزالة هذه المنطقة المستهدفة يتم إعادة توصيل نهايات القطع ويلاحظ الآثار على الخلايا. وبالتالي تم تعديل نظام كريسبر Cas9 من آلية بكتيرية.ويمكن أن تستخدم لمجموعة من تقنيات تحرير الجينات.

Thurtle ‐ Schmidt و Deborah M. و Te-Wen Lo. "البيولوجيا الجزيئية في الطليعة: مراجعة لتحرير الجينات CRISPR / CAS9 للطلاب الجامعيين". الكيمياء الحيوية وتعليم البيولوجيا الجزيئية 46 ، العدد 2 (2018): 195-205. [<a href = "https://doi.org/10.1002/bmb.21108" target = "__blank" >المصدر</a>]

لاندر ، إريك س. "أبطال كريسبر." زنزانة 164 ، رقم 1 (14 يناير 2016): 18-28. [<a href = "https://doi.org/10.1016/j.cell.2015.12.041" target = "__blank" >المصدر</a>]

كريسبر crispr

تسمح تقنيات تحرير الجينوم للعلماء بتعديل الحمض النووي للكائن الحي عن طريق إضافة أو إزالة أو إعادة ترتيب المواد الجينية في مواقع جينية محددة. يمكن استخدام هذه الأنواع من التقنيات لعلاج الاضطرابات الوراثية مثل الهيموفيليا وفقر الدم المنجلي. يعد نظام كريسبر-كاس٩ (CRISPR-Cas9) أحد أدوات البحث الشائعة والمستخدمة على نطاق واسع في مجال تحرير الحمض النووي والتي يمكن أن تؤدي إلى علاجات آمنة وفعالة للاضطرابات الوراثية. يرمز كريسبر-كاس٩ إلى التكرارات المتناظرة القصيرة المنتظمة المتباعدة بانتظام والبروتين ٩ المرتبط بـكريسبر. يتكون نظام كريسبر-كاس٩ الأساسي من نوكلياز كاس٩ داخلي و حمض ريبي صغير يوجه كاس٩ إلى الحمض النووي المستهدف.

<h4>المنشا</h4>

تم ملاحظة تسلسلات كريسبر لأول مرة في البكتيريا و تم تحديدها لاحقاً في العتائق. اكتشف الباحثون أن نظام كريسبر-كاس٩ يقدم دفاعاً مناعياً تكيفياً ضد غزو الفيروسات. تلتقط العديد من البكتيريا ومعظم العتائق تسلسلات قصيرة من الحمض النووي الفيروسي لإنشاء مكتبة من شرائح الحمض النووي للفيروس ، أو مصفوفات كريسبر. عندما يتم إعادة تعريض بدائيات النوى لنفس الفيروس أو فئة الفيروسات ، تُستخدم مصفوفات كريسبر لنسخ مقاطع الحمض النووي الريبي الصغيرة التي تساعد في التعرف على الغزاة الفيروسيين ومن ثم تدمير الحمض النووي الفيروسي باستخدام كاس٩ أو نوكلياز داخلي مماثل.

<h4>استخدام تقنية كريسبر-كاس٩ (CRISPR-Cas9)</h4>

يستخدم كريسبر-كاس٩ بشكل شائع في المختبر لإزالة الحمض النووي وإدخال تسلسل حمض نووي جديد في مكانه. لتحقيق ذلك ، يجب على الباحثين أولاً إنشاء جزء صغير من الحمض النووي الريبي يسمى الحمض الريبي الدليل ، مع تسلسل قصير يسمى تسلسل الدليل الذي يرتبط بتسلسل هدف محدد على الحمض النووي الجيني. يمكن أن يرتبط الحمض الريبي الدليل أيضاً بـ كاس٩ (أو نوكليازات داخلية أخرى مثل Cpf1). يتم إعطاء الحمض الريبي الدليل وبروتين كاس٩ إلى خلية ذات أهمية حيث يحدد الحمض الريبي الدليل تسلسل الحمض النووي المستهدف ويقوم كاس٩ بشقها.

تقوم آلية الخلية بعد ذلك بإصلاح الخيوط المكسورة عن طريق إدخال أو حذف نيوكليوتيدات عشوائية ، مما يجعل الجين المستهدف غير نشط. بدلاً من ذلك ، يمكن إدخال تسلسل حمض نووي مخصص في الخلية جنباً إلى جنب مع الحمض الريبي الدليل و كاس٩ ، والذي يعمل كقالب لآلة الإصلاح ويحل محل التسلسل المقتطع. هذه طريقة فعالة للغاية للباحثين لتعطيل ”knock out“ جين لدراسة آثاره أو استبدال جين متحور بنسخة طبيعية على أمل علاج المرض.

<h4>الاعتبارات الأخلاقية و الجدوى في البشر</h4>

نتيجة لقدرات تعديل الجينات الهامة لنظام كريسبر-كاس٩ ، كان هناك جدل كبير حول استخدامه ، خاصة فيما يتعلق بتحرير الأجنة. ادعى عالم صيني مؤخراً أنه ابتكر أطفالاً ذوي جينوم معدل باستخدام تقنية كريسبر لتعطيل الجين المتورط في عدوى فيروس نقص المناعة البشرية. أدى ذلك إلى احتجاج عالمي من العلماء المهتمين بالاعتبارات الأخلاقية وسلامة الإجراءات. وصف الكثيرون هذه الخطوة بأنها سابقة لأوانها ، وأعرب آخرون عن مخاوفهم بشأن التأثيرات الجينومية غير المستهدفة. في حين أن عدد تطبيقات التكنولوجيا الحيوية الممكنة لنظام كريسبر-كاس٩ متعدد ، فمن المهم النظر في التحديات المستقبلية التي قد تنشأ نتيجة لاستخدامه.

كريسبر CRISPR

تسمح تقنيات تحرير الجينوم للعلماء بتعديل الحمض النووي للكائن الحي عن طريق إضافة أو إزالة أو إعادة ترتيب المواد الوراثية في ...

Education

JoVE Core

علم الأحياء

Genetics

علم الوراثة

Biotechnology

التكنولوجيا الحيوية

KEY TERMS AND CONCEPTS

المصطلحات والمفاهيم الأساسية

crispr-cas9 gene editing in action

CRISPR-Cas9 is a genome editing tool that lets scientists change DNA at a specific location. It can add, remove, or rearrange genetic material. Researchers study it because it may help treat genetic disorders such as hemophilia and sickle cell anemia.

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats, and Cas9 is a CRISPR-associated protein 9. The basic system includes a Cas9 endonuclease, which is an enzyme that cuts DNA, and a small RNA that guides Cas9 to the target sequence. Together, they make CRISPR-Cas9 a precise research tool for DNA editing.

CRISPR sequences were first seen in bacteria and later found in archaea. Scientists discovered that this system acts like an adaptive immune defense against viruses. Many bacteria and most archaea store short pieces of viral DNA in CRISPR arrays. When the same virus returns, these RNA segments help identify it, and Cas9 or a similar endonuclease destroys the viral DNA.

In the lab, CRISPR-Cas9 is often used to remove DNA and replace it with a new sequence. Researchers first design a guide RNA, which contains a guide sequence that matches a target sequence on genomic DNA. The guide RNA and Cas9 are then placed in the cell of interest. The guide RNA finds the target, Cas9 cuts the DNA, and the cell repairs the break by adding or deleting random nucleotides, which can turn the gene off.

Scientists can also add a custom DNA template with the guide RNA and Cas9. The repair machinery can use that template to replace the removed sequence. This makes CRISPR useful for gene knockouts and for replacing a mutated gene with a normal copy in hopes of treating disease. Other endonucleases, such as Cpf1, can also work with guide RNA.

CRISPR-Cas9 has also raised major ethical and safety questions, especially about embryo editing. A Chinese scientist claimed to have used CRISPR to create genome-edited babies by disabling a gene linked to HIV infection. That claim led to global concern because many scientists thought the work was premature and worried about off-target genomic effects. CRISPR has many possible biotech uses, but future challenges still need careful attention.

CRISPR-Cas9 Gene Editing in Action

what is genetic engineering?

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crispr

CRISPR-Cas9 is a genome editing tool that lets scientists change DNA at a specific location. It can add, remove, or rearrange genetic material. Researchers study it because it may help treat genetic disorders such as hemophilia and sickle cell anemia. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats, and Cas9 is a CRISPR-associated protein 9. The basic system includes a Cas9 endonuclease, which is an enzyme that cuts DNA, and a small RNA that guides Cas9 to the...

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genomics

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key terms and concepts

Learn how CRISPR-Cas9 edits DNA, uses guide RNA and Cas9, and why scientists debate its use in embryos and gene therapy.

CRISPR-Cas9 Gene Editing in Action | JoVE K12

JoVE Core series brings biology to life through over 300 concise and easy-to-understand animated video lessons that explain key concepts in biology, plus more than 150 scientist-in-action videos that show actual research experiments conducted in today’s laboratories.

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BIO1.ETS2.2

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An Introduction to Stem Cell Biology

Cells that can differentiate into a variety of cell types, known as stem cells, are at the center of one of the most exciting fields of science today. ...

embryonic-stem-cell-culture-and-differentiation

Embryonic Stem Cell Culture and Differentiation

Culturing embryonic stem (ES) cells requires conditions that maintain these cells in an undifferentiated state to preserve their capacity for self-renewal ...

Induced Pluripotency

Induced pluripotent stem cells (iPSCs) are somatic cells that have been genetically reprogrammed to form undifferentiated stem cells. Like embryonic stem ...

An Introduction to Organogenesis

Organogenesis is the process by which organs arise from one of three germ layers during the later stages of embryonic development. Researchers studying ...

tissue-regeneration-with-somatic-stem-cells

Tissue Regeneration with Somatic Stem Cells

Somatic or adult stem cells, like embryonic stem cells, are capable of self-renewal but demonstrate a restricted differentiation potential. Nonetheless, ...

Analyze scientific and ethical arguments to support the pros and cons of applications of a specific biotechnology technique such as stem cell usage, in vitro fertilization, or genetically modified organisms.
EXPLANATION: The utilization of new technologies in any field of science is dependent on both economic and social factors. In addition to evaluating these factors, scientists must also consider long-term consequences that may not be initially apparent. The emphasis should be on the construction of a rationale argument that supports a position on the use of an application with ethical and social impact. Students should begin to appreciate the differences in ethical values that exist, and recognize that discussion of these values is imperative as knowledge and technology continue to advance, even when resolutions of differences can be rare. After investigation of a specific biotechnology application, students can write a position paper and/or participate in a classroom debate.

CRISPR-Cas9 Gene Editing in Action

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