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

links-among-engineering-technology-science-and-society

Links Among Engineering, Technology, Science, and Society

BIO1.ETS2.3

CRISPR

System CRISPR-Cas9 to narzędzie do edycji DNA, które oznacza Clustered Regularly Interspaced Short Palindromic Repeats i CRISPR Associated Protein 9.

Po raz pierwszy zaobserwowany u bakterii, CRISPR-Cas9 jest środkiem obrony przed wirusami. Gdy obce wirusowe DNA dostaje się do bakterii, jest przetwarzane na mniejsze fragmenty, które mogą zostać wstawione do regionu genomu bakteryjnego zwanego CRISPR Locus.

Kiedy region jest transkrybowany, produkt łączy się z mniejszymi RNA zwanymi tracrRNA, które mogą pomóc w orientacji zarówno białka Cas9, jak i RNAzy na cząsteczkę. Ten ostatni z nich rozszczepia transkrypcję.

Efektem końcowym jest kilka kompleksów, z których każdy składa się z białka Cas9, tracrRNA i crRNA pochodzącego z DNA w Locus. RNA CRISPR w tych strukturach rozpoznaje i prowadzi Cas9 do wirusowego DNA, które jest następnie rozszczepiane i niszczone.

Naukowcy wykorzystują CRISPR-Cas9 poprzez syntezę pojedynczych cząsteczek RNA, które naśladują tracrRNA i CRISPR RNA, które mogą celować w interesujący gen. Na przykład, gdy dwa takie przewodnikowe RNA są wprowadzane do komórek z Cas9 i oba celują w ten sam gen, sekwencja może zostać wycięta.

Po usunięciu tego obszaru docelowego odcięte końce są ponownie łączone i obserwuje się wpływ na komórki.

W ten sposób system CRISPR-Cas9 jest modyfikowany na podstawie mechanizmu bakteryjnego. I może być stosowany do szeregu technik edycji genów.

Thurtle-Schmidt, Deborah M. i Te‐Wen Lo. "Biologia molekularna w czołówce: przegląd edycji genów CRISPR/CAS9 dla studentów". Edukacja w zakresie biochemii i biologii molekularnej 46, nr 2 (2018): 195–205. [<a href="https://doi.org/10.1002/bmb.21108" target="__blank">Źródło</a>]

Lander, Eric S. "Bohaterowie CRISPR." Komórka 164, nr 1 (14 stycznia 2016): 18–28. [<a href="https://doi.org/10.1016/j.cell.2015.12.041" target="__blank">Źródło</a>]

crispr

Technologie edycji genomu umożliwiają naukowcom modyfikowanie DNA organizmu poprzez dodawanie, usuwanie lub rearanżację materiału genetycznego w określonych lokalizacjach genomu. Tego typu techniki można potencjalnie zastosować w leczeniu chorób genetycznych, takich jak hemofilia i anemia sierpowatokrwinkowa. Jednym z popularnych i szeroko stosowanych narzędzi badawczych do edycji DNA, które może prowadzić do bezpiecznych i skutecznych leków na choroby genetyczne, jest system CRISPR-Cas9. CRISPR-Cas9 oznacza Clustered Regularly Interspaced Short Palindromic Repeats i białko 9 związane z CRISPR. Podstawowy system CRISPR-Cas9 składa się z endonukleazy Cas9 i małego RNA, który kieruje Cas9 do docelowego DNA.

<h4>Pochodzenie</h4>

Sekwencje CRISPR po raz pierwszy zaobserwowano u bakterii, a później zidentyfikowano u archeonów. Naukowcy odkryli, że system CRISPR-Cas9 zapewnia adaptacyjną obronę immunologiczną przed inwazją wirusów. Wiele bakterii i większość archeonów wychwytuje krótkie sekwencje wirusowego DNA, tworząc bibliotekę segmentów DNA wirusa, czyli macierze CRISPR. Kiedy prokarioty są ponownie wystawione na działanie tego samego wirusa lub klasy wirusów, do transkrypcji małych segmentów RNA stosuje się macierze CRISPR, które pomagają rozpoznać wirusowych najeźdźców, a następnie zniszczyć wirusowy DNA za pomocą Cas9 lub podobnej endonukleazy.

<h4>Korzystanie z technologii CRISPR-Cas9</h4>

CRISPR-Cas9 jest powszechnie stosowany w laboratorium do usuwania DNA i wstawiania w jego miejsce nowej sekwencji DNA. Aby to osiągnąć, badacze muszą najpierw stworzyć mały fragment RNA zwany RNA kierującym, z krótką sekwencją zwaną sekwencją kierującą, która wiąże się z określoną sekwencją docelową w genomowym DNA. Przewodnik RNA może również łączyć się z Cas9 (lub innymi endonukleazami, takimi jak Cpf1). Kierujący RNA i białko Cas9 podaje się do interesującej komórki, gdzie kierujący RNA identyfikuje docelową sekwencję DNA, a Cas9 ją rozcina.

Maszyna komórkowa naprawia następnie uszkodzone nici poprzez wstawienie lub usunięcie losowych nukleotydów, czyniąc docelowy gen nieaktywnym. Alternatywnie, do komórki można wprowadzić dostosowaną sekwencję DNA wraz z kierującym RNA i Cas9, która służy jako matryca dla mechanizmu naprawczego i zastępuje wyciętą sekwencję. Jest to dla badaczy bardzo skuteczny sposób “wybijania“ genu w celu zbadania jego skutków lub zastąpienia zmutowanego genu normalną kopią w nadziei na wyleczenie choroby.

<h4>Rozważania etyczne i wykonalności u ludzi</h4>

W wyniku znaczących możliwości modyfikacji genów systemu CRISPR-Cas9 toczyła się wielka debata na temat jego zastosowania, szczególnie w odniesieniu do edycji zarodków. Chiński naukowiec oświadczył niedawno, że stworzył dzieci ze zmodyfikowanym genomem przy użyciu technologii CRISPR w celu wyłączenia genu odpowiedzialnego za zakażenie wirusem HIV. Doprowadziło to do ogólnoświatowego protestu naukowców zaniepokojonych względami etycznymi i bezpieczeństwem procedury. Wielu określiło to posunięcie jako przedwczesne, a inni wyrazili obawy związane z efektami genomicznymi odbiegającymi od docelowych. Chociaż liczba możliwych zastosowań biotechnologicznych systemu CRISPR-Cas9 jest duża, ważne jest, aby wziąć pod uwagę przyszłe wyzwania, które mogą pojawić się w wyniku jego wykorzystania.

Technologie edycji genomu pozwalają naukowcom modyfikować DNA organizmu poprzez dodawanie, usuwanie lub przegrupowywanie materiału genetycznego w określonym ...

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Biologia

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Genetyka

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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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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

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genomics

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Genomics studies the full set of genetic material in an organism. In humans, that genome includes the DNA carried in 23 pairs of chromosomes in the nucleus, along with mitochondrial DNA. Genomics looks at both coding DNA and non-coding DNA, which helps scientists study how living things are built and how they differ. This field is useful in many areas of biology and medicine. Scientists use genomics to build phylogenetic trees, improve crop productivity and sustainability, help with criminal...

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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Obtain, evaluate, and communicate information on how molecular biotechnology may be used in a variety of fields.
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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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