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

The CRISPR-Cas9 system is a DNA editing tool that stands for, Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR associated protein 9.

First observed in bacteria, CRISPR-Cas9 is a means of defense against viruses. As foreign viral DNA enters a bacterium, it&#39;s processed into smaller fragments, which may be inserted into a region of the bacterial genome called a CRISPR Locus.

When the region is transcribed, the product associates with smaller RNAs called tracrRNAs which may help to orient both the Cas9 protein and RNAse to the molecule. The latter of which cleaves the transcript.

The end result is several complexes each consisting of a Cas9 protein, tracrRNA and a crRNA derived from DNA in the Locus. The CRISPR RNA in these structures recognizes and guides Cas9 to viral DNA which is then cleaved and destroyed.

Scientists harness CRISPR-Cas9 by synthesizing individual RNA molecules that mimic tracrRNA and CRISPR RNA which can target a gene of interest. For example when two such guide RNAs are introduced into cells with Cas9 and both target the same gene, a sequence can be excised.

Once this target region is removed the cut ends are reconnected and the effects on the cells are observed.

Thus the CRISPR-Cas9 system is modified from a bacterial mechanism. And can be employed for an array of gene editing techniques.

crispr

Genome editing technologies allow scientists to modify an organism&rsquo;s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9. A basic CRISPR-Cas9 system consists of a Cas9 endonuclease and a small RNA that guides Cas9 to the target DNA.

<h4>Origin</h4>

CRISPR sequences were first observed in bacteria and later identified in archaea. Researchers discovered that the CRISPR-Cas9 system serves an adaptive immune defense against invading viruses. Many bacteria and most archaea capture short sequences of the viral DNA to create a library of virus DNA segments, or CRISPR arrays. When the prokaryotes are re-exposed to the same virus or class of viruses, CRISPR arrays are used to transcribe small RNA segments that help recognize viral invaders and subsequently destroy viral DNA with Cas9 or a similar endonuclease.

<h4>Using CRISPR-Cas9 Technology</h4>

CRISPR-Cas9 is commonly used in the laboratory to remove DNA and insert a new DNA sequence in its place. To achieve this, researchers must first create a small fragment of RNA called the guide RNA, with a short sequence called the guide sequence that binds to a specific target sequence on genomic DNA. The guide RNA can also associate with Cas9 (or other endonucleases like Cpf1). The guide RNA and Cas9 protein are administered to a cell of interest where the guide RNA identifies the target DNA sequence and Cas9 cleaves it.

The cell&rsquo;s machinery then repairs the broken strands by inserting or deleting random nucleotides, rendering the target gene inactive. Alternatively, a customized DNA sequence may be introduced into the cell along with the guide RNA and Cas9, that serves as a template for the repair machinery and replaces the excised sequence. This is a highly effective way for researchers to &ldquo;knock out&rdquo; a gene to study its effects or replace a mutated gene with a normal copy in hopes of curing a disease.

<h4>Ethical and Feasibility Considerations in Humans</h4>

As a result of the significant gene modification capabilities of the CRISPR-Cas9 system, there has been great debate over its use, especially in regards to embryo editing. A Chinese scientist recently claimed to have created genome-edited babies using CRISPR technology to disable a gene involved in HIV infection. This led to a global outcry from scientists concerned about the ethical and safety considerations of the procedure. Many have called the move premature, and others have expressed concerns over off-target genomic effects. While the number of possible biotech applications for the CRISPR-Cas9 system is numerous, it is important to consider future challenges that may arise as a result of its use.

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

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KEY TERMS AND CONCEPTS

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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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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