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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Biology

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Links Among Engineering, Technology, Science, and Society

BIO1.ETS2.3

CRISPR

- [Narratore] Il sistema
CRISPR-Cas9 è uno strumento di

modifica del DNA che
consente di raggruppare

brevi ripetizioni a
intervalli regolari, e CRISPR

associate a proteine 9.
Prima osservato nei batteri,

il CRISPR-Cas9 è un mezzo
di difesa contro i virus.

Come il DNA virale estraneo
entra in un batterio,

è processato in frammenti più piccoli,

che possono essere inseriti in una regione

del genoma batterico
chiamata locus CRISPR.

Quando la regione viene
trascritta, il prodotto si associa

con RNA più piccoli chiamati tracrRNAs,

che può aiutare ad orientare
sia la proteina Cas9

sia la RNAse alla molecola,
l'ultima della quale

divide la trascrizione.

Il risultato finale è
di diversi complessi,

ciascuno costituito da una
proteina Cas9, tracrRNA,

e un CRISPR-RNA, derivato
dal DNA nel locus.

Il CRISPR-RNA in queste strutture

riconosce e guida il Cas9 al DNA virale,

che viene poi diviso e distrutto.

Gli scienziati sfruttano CRISPR-Cas9

sintetizzando singole molecole di RNA

che imitano tracrRNA e crRNA,

che possono colpire un gene di interesse.

Ad esempio, quando due di questi RNA guida

vengono introdotti nelle
cellule con Cas9, ed entrambi

mirano allo stesso gene, una
sequenza può essere eliminata.

Una volta che questa regione
target viene rimossa,

le estremità tagliate sono ricollegate,

e gli effetti sulle
cellule vengono osservati.

Pertanto, il sistema
CRISPR-Cas9 viene modificato

da un meccanismo batterico
e può essere impiegato

per una serie di tecniche
di modifica dei geni.

Thurtle-Schmidt, Deborah M. e Te-Wen Lo. "Biologia molecolare all'avanguardia: una revisione sull'editing genetico CRISPR/CAS9 per gli studenti universitari". Educazione alla biochimica e alla biologia molecolare 46, n. 2 (2018): 195–205. [<a href="https://doi.org/10.1002/bmb.21108" target="__blank">Sorgente</a>]

Lander, Eric S. "Gli eroi di CRISPR". Cella 164, n. 1 (14 gennaio 2016): 18–28. [<a href="https://doi.org/10.1016/j.cell.2015.12.041" target="__blank">Sorgente</a>]

crispr

Le tecnologie di editing del genoma consentono agli scienziati di modificare il DNA di un organismo mediante l'aggiunta, la rimozione o il riarrangiamento di materiale genetico in posizioni genomiche specifiche. Questi tipi di tecniche potrebbero essere potenzialmente utilizzati per curare disturbi genetici come l'emofilia e l'anemia falciforme. Uno strumento di ricerca di editing del DNA popolare e ampiamente usato che potrebbe portare a cure sicure ed efficaci per i disturbi genetici è il sistema CRISPR-Cas9. CRISPR-Cas9 è l'acronimo di Clustered Regularly Interspaced Short Palindromic Repeats e proteina CCR 9. Un sistema CRISPR-Cas9 di base è costituito da un endonucleasi Cas9 e un piccolo RNA che guida Cas9 verso il DNA di destinazione.

<h4>Origine</h4>

Le sequenze CRISPR sono state osservate per la prima volta nei batteri e successivamente identificate negli archei. I ricercatori hanno scoperto che il sistema CRISPR-Cas9 serve una difesa immunitaria adatta contro l'invasione dei virus. Molti batteri e la maggior parte degli archei catturano brevi sequenze del DNA virale per creare una libreria di segmenti di DNA virus, o array CRISPR. Quando i procarioti vengono riesposti allo stesso virus o classe di virus, i vettori CRISPR vengono utilizzati per trascrivere piccoli segmenti di RNA che aiutano a riconoscere gli invasori virali e successivamente a distruggere il DNA virale con Cas9 o un endonucleasi simile.

<h4>Utilizzo della tecnologia CRISPR-Cas9</h4>

CRISPR-Cas9 è comunemente usato in laboratorio per rimuovere il DNA e inserire una nuova sequenza di DNA al suo posto. Per raggiungere questo obiettivo, i ricercatori devono prima creare un piccolo frammento di RNA chiamato RNA guida, con una breve sequenza chiamata sequenza di guida che si lega a una sequenza bersaglio specifica sul DNA genomico. La guida RNA può anche associarsi a Cas9 (o altre endonucleasi come Cpf1). La guida RNA e proteina Cas9 vengono somministrate a una cellula di interesse dove l'RNA guida identifica la sequenza di DNA bersaglio e Cas9 la fende.

Il macchinario della cellula ripara quindi i fili rotti inserendo o eliminando nucleotidi casuali, rendendo inattivo il gene bersaglio. In alternativa, una sequenza di DNA personalizzata può essere introdotta nella cellula insieme alla guida RNA e Cas9, che funge da modello per i macchinari di riparazione e sostituisce la sequenza eccitata. Questo è un modo molto efficace per i ricercatori di "eliminare" un gene per studiarne gli effetti o sostituire un gene mutato con una copia normale nella speranza di curare una malattia.

<h4>Considerazioni etiche e di fattibilità negli esseri umani</h4>

Come risultato delle significative capacità di modificazione genica del sistema CRISPR-Cas9, c'è stato un grande dibattito sul suo utilizzo, soprattutto per quanto riguarda l'editing degli embrioni. Uno scienziato cinese ha recentemente affermato di aver creato bambini che hanno curato il genoma utilizzando la tecnologia CRISPR per disabilitare un gene coinvolto nell'infezione da HIV. Ciò ha portato a una protesta globale da parte di scienziati preoccupati per le considerazioni etiche e di sicurezza della procedura. Molti hanno definito prematura la mossa, mentre altri hanno espresso preoccupazioni per gli effetti genomici fuori bersaglio. Sebbene il numero di possibili applicazioni biotecnologiche per il sistema CRISPR-Cas9 sia numeroso, è importante considerare le sfide future che potrebbero sorgere a causa del suo utilizzo.

Genome editing technologies allow scientists to modify an organism&#8217;s DNA via the addition, removal, or rearrangement of genetic material at specific ...

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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

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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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