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

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BIO1.ETS2.3

CRISPR

Система CRISPR-Cas9 это инструмент редактирования ДНК, который дословно обозначает Кластерные регулярно чередующиеся короткие палиндромные повторы, и белок, ассоциированный с CRISPR 9. Впервые найденный у бактерий, CRISP-Cas9 это средство защиты от вирусов. Вирусная ДНК, как инородная, попадает в бактерию, она обрабатывается в небольшие фрагменты, которые могут быть включены в регион бактериального генома, локус CRISPR.Когда регион расшифрован, Продукт, который ассоциируется с небольшими РНК, называемыми тракрРНК, которые могут помочь сориентировать обоих, Белок Cas9 и. RNase к молекуле, и последний из них разъединяет транскрипт. Конечный результат несколько комплексов, каждый из них состоит из протеина Cas9, TracrRNA и CRISPR RNA, полученных от ДНК в локусе.CRISPR РНК в этих структурах узнаёт и направляет Cas9 к вирусной ДНК, которая затем расщепляется и уничтожается. Ученые используют CRISPR-Cas9 Синтезируя индивидуальные молекулы РНК, Которые имитируют РНК трарРНК и РНК CRISPR, и которые могут быть нацелены на интересующий ген. Например, если две таких направляющих РНК внедрены в клетки с Cas9, и обе они нацелены на один и тот же ген, последовательность может быть вырезана.После удаления целевой области концы обрезки снова присоединены, и наблюдается влияние на клетки. Таким образом, система CRISPR-Cas9 модифицирована из бактериального механизма, и может быть использована для множества методов редактирования генов.

Тертл-Шмидт, Дебора М. и Те-Вен Ло. «Молекулярная биология на переднем крае: обзор редактирования генов CRISPR/CAS9 для студентов». Образование в области биохимии и молекулярной биологии 46, No 2 (2018): 195–205. [<a href="https://doi.org/10.1002/bmb.21108" target="__blank">Источник</a>]

Ландер, Эрик С. «Герои CRISPR». Cell 164, No1 (14 января 2016 г.): 18–28. [<a href="https://doi.org/10.1016/j.cell.2015.12.041" target="__blank">Источник</a>]

crispr

Технологии редактирования генома позволяют ученым изменять ДНК организма путем добавления, удаления или перестановки генетического материала в определенных участках генома. Эти типы методов потенциально могут быть использованы для лечения генетических расстройств, таких как гемофилия и серповидно-клеточная анемия. Одним из популярных и широко используемых инструментов исследования редактирования ДНК, который может привести к безопасным и эффективным лекарствам от генетических расстройств, является система CRISPR-Cas9. CRISPR-Cas9 означает кластерные регулярно межпространственные короткие палиндромные повторы и связанный с CRISPR белок 9. Базовая система CRISPR-Cas9 состоит из эндонуклеазы Cas9 и небольшой РНК, которая направляет Cas9 к ДНК цели.

<h4>Происхождения</h4>

CrispR последовательности были впервые замечены в бактериях, а затем определены в археях. Исследователи обнаружили, что система CRISPR-Cas9 служит адаптивной иммунной защитой от вторжения вирусов. Многие бактерии и большинство археев захвата короткие последовательности вирусной ДНК для создания библиотеки сегментов ДНК вируса, или CRISPR массивов. Когда прокариоты повторно подвергаются воздействию одного и того же вируса или класса вирусов, массивы CRISPR используются для транскрибирования небольших сегментов РНК, которые помогают распознавать вирусных захватчиков, а затем уничтожить вирусную ДНК с Cas9 или аналогичной эндонуклеазой.

<h4>Использование технологии CRISPR-Cas9</h4>

CRISPR-Cas9 обычно используется в лаборатории для удаления ДНК и вставки новой последовательности ДНК на ее месте. Для достижения этой цели исследователи должны сначала создать небольшой фрагмент РНК, называемый направляющей РНК, с короткой последовательностью, называемой последовательностью руководства, которая связывается с определенной целевой последовательностью геномной ДНК. Руководство РНК может также ассоциироваться с Cas9 (или других эндонуклейсов, как Cpf1). Руководство РНК и Cas9 белка вводятся в ячейку, представляющие интерес, где руководство РНК определяет целевую последовательность ДНК и Cas9 расщепляет его.

Затем механизм клетки восстанавливает сломанные цепочки, внося или удаляя случайные нуклеотиды, делая целевой ген неактивным. Кроме того, в ячейку может быть введена настраиваемая последовательность ДНК вместе с направляющий РНК и Cas9, который служит шаблоном для ремонтного оборудования и заменяет вырезанную последовательность. Это очень эффективный способ для исследователей, чтобы "выбить" ген для изучения его эффекта или заменить мутировавший ген нормальной копией в надежде вылечить болезнь.

<h4>Этические и технико-экономические соображения в людях</h4>

В результате значительных возможностей модификации генов системы CRISPR-Cas9, были большие дебаты по поводу ее использования, особенно в отношении редактирования эмбрионов. Китайский ученый недавно заявил, что создал геном-редактируемых младенцев с использованием технологии CRISPR, чтобы отключить ген, участвующий в ВИЧ-инфекции. Это вызвало глобальный протест со стороны ученых, обеспокоенных этическими соображениями и соображениями безопасности процедуры. Многие назвали этот шаг преждевременным, а другие выразили озабоченность по поводу внецелевременных геномных эффектов. Хотя количество возможных биотехнологических приложений для системы CRISPR-Cas9 многочисленны, важно учитывать будущие проблемы, которые могут возникнуть в результате ее использования.

Технологии редактирования генома позволяют ученым модифицировать ДНК организма путем добавления, удаления или перегруппировки генетического материала в определенные ...

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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?

How Scientists Edit DNA for New Traits

Genetic engineering changes an organism’s DNA to add useful traits. Scientists use it in bacteria, plants, animals, and even viruses for research, medicine, agriculture, and industry. It can bring major benefits, but it also raises ethical concerns about humans and the food supply. This work is possible because DNA has the same basic structure and genetic code across life. That shared code lets scientists modify DNA in several ways. One method is CRISPR/Cas9, often called molecular scissors,...

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recombinant dna technology

Recombinant DNA in Cloning and GMOs

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PCR Cycle for DNA Amplification

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complementary dna

From mRNA to cDNA for Gene Studies

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antibiotic selection

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transgenic organisms

Creating Transgenic Plants and Animals

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in-vitro mutagenesis (gene knockouts)

Knockout Mice and Gene Replacement

Knockout mice help researchers study gene function by showing what happens when a gene is turned off. These engineered animals are also useful for modeling human diseases such as cancer, Parkinson’s disease, and diabetes. A specific gene can be targeted by using a targeting vector, which is a piece of engineered DNA that replaces the normal gene and inactivates it. The vector has matching DNA sequences on both ends. These homologous sequences let the vector swap in for the target gene through...

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...

gene therapy

Gene Delivery for Serious Diseases

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adult stem cells

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reproductive cloning

Cloning Animals with SCNT

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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

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Genetic engineering &#8211; the process of purposefully altering an organism&#8217;s DNA &#8211; has been used to create powerful research tools and model ...

Recombineering and Gene Targeting

One of the most widely used tools in modern biology is molecular cloning with restriction enzymes, which create compatible ends between DNA fragments that ...

Genome Editing

A well-established technique for modifying specific sequences in the genome is gene targeting by homologous recombination, but this method can be ...

Recombinant DNA

DNA Ligation Reactions

In molecular biology, ligation refers to the joining of two DNA fragments through the formation of a phosphodiester bond.  An enzyme known as a ligase ...

Restriction Enzyme Digests

Restriction enzymes or endonucleases recognize and cut DNA at a specific sequence.  These enzymes occur naturally in bacteria as a defense against ...

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Restriction Enzymes

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bacterial-transformation-the-heat-shock-method

Bacterial Transformation: The Heat Shock Method

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Maxam-Gilbert Sequencing

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Obtain, evaluate, and communicate information on how molecular biotechnology may be used in a variety of fields.
EXPLANATION: The goal is to help students appreciate that the basic science knowledge being studied can be manipulated and used to design useful tools for further scientific investigations, medical treatments, agricultural yields, etc. A detailed understanding of any technical procedures is not expected, but rather a "big picture" view of concepts the technology utilizes and the applications the technology is being used for. These technologies may be best investigated along with the standards from LS1 From Molecules to Organisms: Structure and Functions, as the students are learning about DNA and protein. Comparative DNA and protein sequence analysis is also a large part of current phylogenic research and could accompany LS4 Biological Change: Unity and Diversity. Students may also consider the pros and cons of biotechnical applications. Investigation of techniques utilized could come from field trips to witness the use of these techniques in practice, lab exercises, virtual labs, simulation activities, interviews with professionals, etc. Molecular techniques could include: PCR, electrophoresis, restriction enzyme digestion of DNA, DNA sequencing, plasmid-based transformation, transfection, etc. These techniques are used in fields of medicine, agriculture, biomedical engineering, forensic science, etc.

BIO1.ETS2.1

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Investigate means by which karyotypes are utilized in diagnostic medicine.
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BIO1.ETS2.2

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Reproductive cloning is the process of producing a genetically identical copy&mdash;a clone&mdash;of an entire organism. While clones can be produced by ...

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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