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

- [Narrateur] Le système CRISPR-Cas9est un outil d'édition d'ADN. 
qui signifie répétitions palindromiquesgroupées, courtes régulièrement espacéeset la protéine 9 associée au CRISPR. 
D'abord observé chez les bactéries, CRISPR-Cas9est un moyen de défense contre les virus. 
Lorsque de l'ADN viral étranger pénètre dans une bactérie,il est transformé en fragments plus petits,qui peuvent être insérés dans une régiondu génome bactérien appelée locus CRISPR. 
Lorsque la région est transcrite, le produit s'associe àdes ARN plus petits appelés tracrARN,qui peuvent aider à orienter à la fois la protéine Cas9et l'ARNase vers la molécule, cette dernièrequi scinde le transcrit. 
Le résultat final donne plusieurs complexes,chacun consistant en une protéine Cas9, un tracrARN,et un ARN CRISPR, dérivés de l'ADN dans le locus. 
L'ARN CRISPR dans ces structuresreconnaît et guide Cas9 vers l'ADN viral,qui est ensuite scindé et détruit. 
Les scientifiques exploitent le CRISPR-Cas9en synthétisant des molécules d'ARN individuellesqui imitent les ARNrcr et ARNt,lesquels peuvent cibler un gène d'intérêt. 
Par exemple, lorsque deux de ces ARN guidessont introduits dans des cellules avec Cas9et qu'ils ciblent tous les deux le même gène,une séquence peut être retranchée. 
Une fois que cette région cible est supprimée,les extrémités coupées sont reconnectéeset les effets sur les cellules sont observés. 
Ainsi, le système CRISPR-Cas9 est modifiéà partir d'un mécanisme bactérien et peut être utilisépour un ensemble de techniques d'édition de gènes.

Thurtle‐Schmidt, Deborah M. et Te’Wen Lo. « Biologie moléculaire à la pointe : une revue sur l’édition de gènes CRISPR/CAS9 pour les étudiants de premier cycle. » Biochimie et biologie moléculaire Enseignement 46, n° 2 (2018) : 195-205. [<a href="https://doi.org/10.1002/bmb.21108 » target="__blank">Source</a>]

Lander, Eric S. « Les héros de CRISPR », Cellule 164, n° 1 (14 janvier 2016) : 18-28. [<a href="https://doi.org/10.1016/j.cell.2015.12.041 » target="__blank">Source</a>]

crispr

Les technologies d’édition du génome permettent aux scientifiques de modifier l’ADN d’un organisme par l’ajout, le retrait ou le réarrangement du matériel génétique à des endroits génomiques spécifiques. Ces types de techniques pourraient potentiellement être utilisés pour guérir des troubles génétiques tels que l’hémophilie et l’anémie drépanocytaire. Dans la recherche, un moyen reconnu et largement utilisé d’éditer de l’ADN qui pourrait mener à des remèdes sûrs et efficaces pour les maladies génétiques est le système CRISPR-Cas9. CRISPR-Cas9 signifie Clustered Regularly Interspaced Short Palindromic Repeats (“ courtes répétitions palindromiques groupées et régulièrement espacées ”) et CRISPR-associated protein 9 (“ protéine 9 associée à CRISPR ”). Un système CRISPR-Cas9 de base se compose d’une endonucléase Cas9 et d’un petit ARN qui guide Cas9 vers l’ADN cible.
 
 <h4>L’origine</h4>
 
 Des séquences de CRISPR ont d’abord été observées chez les bactéries et plus tard identifiées dans les archées. Les chercheurs ont découvert que le système CRISPR-Cas9 sert de défense immunitaire adaptative contre les virus envahissants. De nombreuses bactéries et la plupart des archées capturent de courtes séquences de l’ADN viral pour créer une bibliothèque de segments d’ADN de virus, ou réseaux CRISPR. Lorsque les procaryotes sont réexposés au même virus ou à la même catégorie de virus, les réseaux CRISPR sont utilisés pour transcrire de petits segments d’ARN qui aident à reconnaître les envahisseurs viraux et à détruire par la suite l’ADN viral avec Cas9 ou une endonucléase similaire.
 
 <h4>L’utilisation de la technologie CRISPR-Cas9</h4>
 
 CRISPR-Cas9 est couramment utilisé en laboratoire pour enlever de l’ADN et insérer une nouvelle séquence d’ADN à la place. Pour ce faire, les chercheurs doivent d’abord créer un petit fragment d’ARN appelé ARN guide, avec une courte séquence appelée la séquence de guidage qui se lie à une séquence cible spécifique sur l’ADN génomique. L’ARN guide peut également s’associer à Cas9 (ou d’autres endonucléases comme Cpf1). L’ARN guide et la protéine Cas9 sont administrés à une cellule d’intérêt où l’ARN guide identifie la séquence d’ADN cible et Cas9 la fend.
 
 La machinerie de la cellule répare ensuite les brins cassés en insérant ou en supprimant des nucléotides au hasard, rendant le gène cible inactif. Alternativement, une séquence d’ADN personnalisée peut être introduite dans la cellule avec l’ARN guide et Cas9, qui sert de modèle pour la machinerie de réparation et remplace la séquence excisée. Il s’agit d’un moyen très efficace pour les chercheurs “ d’invalider ” un gène pour étudier ses effets ou de remplacer un gène muté par une copie normale dans l’espoir de guérir une maladie.
 
 <h4>Considérations éthiques et de faisabilité chez l’homme</h4>
 
 En raison des capacités importantes de modification génétique du système CRISPR-Cas9, il y a eu un grand débat sur son utilisation, en particulier en ce qui concerne l’édition d’embryons. Un scientifique chinois a récemment affirmé avoir créé des bébés dont le génome a été édité à l’aide de la technologie CRISPR pour désactiver un gène impliqué dans l’infection par le VIH. Cela a provoqué un tollé mondial de la part des scientifiques préoccupés par les considérations éthiques et la sécurité de la descendance. Beaucoup d’entre eux ont qualifié cette mesure de prématurée, et d’autres ont exprimé des préoccupations au sujet des effets génomiques hors cible. Bien que les applications biotechnologiques possibles pour le système CRISPR-Cas9 soient nombreuses, il est important de tenir compte des défis futurs qui pourraient découler de son utilisation.

Les technologies d’édition du génome permettent aux scientifiques de modifier l’ADN d’un organisme via l’ajout, la suppression ou le réarrangement de matériel génétique à des niveaux spécifiques ...

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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?

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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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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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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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CRISPR-Cas9 Gene Editing in Action

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