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

K12 Standards

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Biology

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Links Among Engineering, Technology, Science, and Society

BIO1.ETS2.3

CRISPR

O sistema CRISPR-Cas9 é uma ferramenta de edição de DNA que significa Repetições Palindrómicas Curtas Agrupadas e Regularmente Interespaçadas, 00:09.850 00:12.733 e a proteína associada a CRISPR 9. Observado pela primeira vez em bactérias, CRISP-Cas9 é um meio de defesa contra vírus. Como estranhos, o DNA viral que entra em uma bactéria, é processado em fragmentos menores, que podem ser inseridos numa região do genoma bacteriano chamado de locus CRISPR.Quando a região é transcrita, o produto associa-se a RNAs menores chamado tracrRNAs, que podem ajudar a orientar ambos a proteína Cas9 e. a RNase à molécula, esta última das quais cliva a transcrição. O resultado final são vários complexos, cada um composto por uma proteína Cas9, TracrRNA e um RNA CRISPR, derivado do DNA no locus.O RNA CRISPR nestas estruturas reconhece e guia Cas9 para o DNA viral, que é então clivado e destruído. Cientistas aproveitam CRISPR-Cas9 sintetizando moléculas de RNA individuais que imitam o tracrRNA e o RNA CRISPR, que podem visar um gene de interesse. Por exemplo, quando dois desses RNAs de guia são introduzidos em células com Cas9, e ambos visam o mesmo gene, é possível excisar uma sequência.Uma vez removida esta região alvo, as extremidades de corte são novamente ligadas, e os efeitos sobre as células são observados. Assim, o sistema CRISPR-Cas9 é modificado a partir de um mecanismo bacteriano, e pode ser empregado para uma série de técnicas de edição de genes.

"Biologia Molecular na Vanguarda: Uma Revisão sobre a Edição de Genes CRISPR / CAS9 para Alunos de Graduação." Educação em Bioquímica e Biologia Molecular 46, nº 2 (2018): 195–205. [<a href="https://doi.org/10.1002/bmb.21108" target="__blank">Fonte</a>]

Lander, Eric S. "Os Heróis do CRISPR." Célula 164, nº 1 (14 de janeiro de 2016): 18–28. [<a href="https://doi.org/10.1016/j.cell.2015.12.041" target="__blank">Fonte</a>]

crispr

As tecnologias de edição de genomas permitem que os cientistas modifiquem o DNA de um organismo através da adição, remoção ou rearranjo de material genético em locais genómicos específicos. Esses tipos de técnicas poderiam potencialmente ser usadas para curar doenças genéticas como hemofilia e anemia falciforme. Uma ferramenta de investigação popular e amplamente utilizada de edição de DNA que poderia levar a curas seguras e eficazes para distúrbios genéticos é o sistema CRISPR-Cas9. CRISPR-Cas9 significa Repetições Palindrómicas Curtas Agrupadas e Regularmente Interespaçadas e Proteína 9 associada a CRISPR. Um sistema CRISPR-Cas9 básico consiste em uma endonuclease Cas9 e um pequeno RNA que guia a Cas9 até o DNA alvo.
 
 <h4>Origem</h4>
 
 As sequências CRISPR foram observadas pela primeira vez em bactérias e posteriormente identificadas em archaea. Investigadores descobriram que o sistema CRISPR-Cas9 serve uma defesa imune adaptativa contra vírus invasores. Muitas bactérias e a maioria das archaea captam sequências curtas do DNA viral para criar uma biblioteca de segmentos de DNA viral, ou matrizes CRISPR. Quando os procariotas voltam a ser expostos ao mesmo vírus ou classe de vírus, as matrizes CRISPR são usadas para transcrever pequenos segmentos de RNA que ajudam a reconhecer invasores virais e, posteriormente, destruir DNA viral com Cas9 ou uma endonuclease semelhante.
 
 <h4>Utilização da Tecnologia CRISPR-Cas9</h4>
 
 Crispr-Cas9 é comumente usada em laboratório para remover DNA e inserir uma nova sequência de DNA no seu lugar. Para isso, os investigadores devem primeiro criar um pequeno fragmento de RNA chamado RNA guia, com uma pequena sequência chamada sequência guia que se liga a uma sequência alvo específica no DNA genómico. O RNA guia também se pode associar à Cas9 (ou a outras endonucleases como a Cpf1). O RNA guia e a proteína Cas9 são administrados a uma célula de interesse onde o RNA guia identifica a sequência de DNA alvo e Cas9 corta-a.
 
 A maquinaria da célula repara então as cadeias quebradas inserindo ou excluindo nucleótidos aleatórios, tornando o gene alvo inativo. Alternativamente, uma sequência de DNA personalizada pode ser introduzida na célula juntamente com o RNA guia e a Cas9, que serve como molde para a maquinaria de reparação e substitui a sequência retirada. Esta é uma maneira altamente eficaz para os investigadores realizarem o “knockout” de um gene para estudar os seus efeitos ou substituir um gene mutante por uma cópia normal na esperança de curar uma doença.
 
 <h4>Considerações Éticas e de Viabilidade em Humanos</h4>
 
 Como resultado das significativas capacidades de modificação genética do sistema CRISPR-Cas9, tem havido um grande debate sobre seu uso, especialmente no que diz respeito à edição de embriões. Um cientista Chinês recentemente afirmou ter criado bebés com genoma editado usando a tecnologia CRISPR para desativar um gene envolvido na infecção pelo HIV. Isso levou a um tumulto global de cientistas preocupados com as considerações éticas e de segurança do procedimento. Muitos chamaram a medida de prematura, e outros expressaram preocupações sobre efeitos genómicos fora do alvo. Embora o número de possíveis aplicações biotecnológicas para o sistema CRISPR-Cas9 seja numeroso, é importante considerar desafios futuros que possam surgir como resultado do seu uso.

As tecnologias de edição do genoma permitem que os cientistas modifiquem o DNA de um organismo por meio da adição, remoção ou rearranjo de material genético em ...

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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,...

dna isolation

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recombinant dna technology

Recombinant DNA in Cloning and GMOs

Recombinant DNA is made by joining DNA from different sources in the lab. Scientists often use it to clone genes and study them in cells that are easy to handle, such as bacteria. Organisms that carry recombinant DNA are called genetically modified organisms, or GMOs. This technology gives scientists new tools for research, medicine, and agriculture. To build recombinant DNA, researchers place a gene of interest into a vector, which is a DNA carrier that moves foreign DNA into a host cell. The...

PCR Cycle for DNA Amplification

PCR is a laboratory method for making many copies of a DNA segment. It can turn a small amount of DNA into millions or even billions of copies in just a few hours. The process uses a heat-resistant DNA polymerase inside a machine called a thermocycler. PCR was developed by Kary Mullis in 1983, and he received the 1993 Nobel Prize in Chemistry for it. The method is fast, relatively inexpensive, and precise. It has become useful for molecular cloning, gene mutagenesis, pathogen detection, gene...

complementary dna

From mRNA to cDNA for Gene Studies

Complementary DNA, or cDNA, is made from messenger RNA (mRNA) so scientists can study which genes are active in cells and tissues. Only genes that are transcribed into mRNA are expressed. By extracting mRNA from cells, researchers can compare gene expression across samples. cDNA is produced by reverse transcription. This process copies mRNA into DNA, which is useful because mRNA does not contain introns, or non-coding regions, and other regulatory sequences. As a result, cDNA can help...

antibiotic selection

Antibiotic Resistance in Cloning

Antibiotic resistance genes help scientists find bacteria that took up a plasmid with a gene of interest. In genetic engineering, this resistance is used as a marker, not just as a medical concern. The same process also connects to the growing problem of resistant bacteria in the real world. Antibiotics, such as penicillin, stop bacterial growth or kill bacteria. When bacteria naturally have, or are engineered to carry, an antibiotic resistance gene, the antibiotic no longer works on them.

transgenic organisms

Creating Transgenic Plants and Animals

Transgenic organisms are genetically engineered to carry a transgene, which is a gene from a different species, in their genome. The inserted gene may be a new gene for that organism or a different version of one it already has. Scientists usually make transgenes with recombinant DNA and DNA cloning methods. Researchers use transgenic bacteria, plants, and animals to answer biological questions and to build useful tools. To make a transgenic organism, they first choose a transgenesis method,...

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

Gene therapy is a technique used to treat serious disease by adding a gene to a person’s cells. The added gene may be a healthy copy of a mutated gene. It may also be a different gene that inactivates or compensates for the disease-causing gene. One example is severe combined immunodeficiency, or SCID, caused by a mutation in the gene for adenosine deaminase. In that case, a working copy of the gene can be inserted so the patient’s cells make the enzyme again. This can cure the disease in some...

embryonic stem cells

How Embryonic Stem Cells Become Specialized

Embryonic stem cells are pluripotent cells that have not yet specialized. Pluripotent means they can give rise to many different cell types in the body. Because of this, they are useful in science and medicine for making cells needed in research or for replacing cells lost to damage or disease. These cells come from the inner cell mass of a blastocyst, which is an early embryo about 3 to 5 days after fertilization in humans. At this stage, the embryo has not yet implanted in the uterus. Human...

adult stem cells

Adult Stem Cells in the Small Intestine

Adult stem cells help maintain tissues by replacing old or damaged cells. These undifferentiated cells divide to make more stem cells or progenitor cells, which then become mature, specialized cells. Small groups of adult stem cells are found in many tissues, including the bone marrow, brain, skin, and gut. The small intestine shows this process clearly. Its lining, called the epithelium, is renewed continuously by adult stem cells. It is also the fastest replaced tissue in the human body,...

induced pluripotent stem cells

Reprogramming Mature Cells into Stem Cells

Reprogramming mature cells into stem cells lets scientists turn specialized cells back into an unspecialized state. These cells are called induced pluripotent stem cells, or iPSCs. Like embryonic stem cells, iPSCs are pluripotent, which means they can form all cell types. To make iPSCs, researchers start with mature cells such as skin fibroblasts or blood cells. The cells are grown in culture, and genes for several transcription factors are delivered with a viral vector. The cells then make...

reproductive cloning

Cloning Animals with SCNT

Reproductive cloning can produce a genetically identical copy of an entire organism. In adult animals, this is usually done with somatic cell nuclear transfer, or SCNT. Another way clones can form is by splitting an early embryo, which is similar to how identical twins arise naturally. In SCNT, an egg cell is taken from an animal and its nucleus is removed. This creates an enucleated egg. A somatic cell, which is any body cell that is not a sex cell, is then taken from the animal to be cloned.

genomics

Genomics and DNA Sequencing

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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A coleção JoVE Core dá vida à biologia através de mais de 300 vídeos de animação concisos e fáceis de entender que explicam conceitos-chave de biologia, além de mais de 150 vídeos de Cientistas em ação que mostram experimentos reais em pesquisa realizados nos laboratórios atualmente.

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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.
EXPLANATION: Engineers incorporate growing scientific knowledge in order to increase benefits to humans and decrease cost and risks. Karyotyping technology and its use in medical diagnosis can complement the standards from LS3: Hereditary: Inheritance, and Variation of Traits, as the students are learning about chromosomal organization of genomic information that determines one's phenotype. Analyze a large set of karyotypes to identify common patterns. Identify variations from the most commonly observed patterns and correlate them with patient phenotypes to propose a cause and effect relationship, discussing limitations of correlative data interpretation.

BIO1.ETS2.2

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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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