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

CRISPR-Cas9システムはDNA編集ツールで 等間隔にスペーサーが入った 短い回文型のリピート配列および CRISPR関連タンパク質9を表わします 最初に細菌で観察されたCRISPR-Cas9は ウイルスに対する防御の手段です 外来ウイルスDNAが細菌に侵入すると より小さな断片に処理され CRISPR遺伝子座と呼ばれる 細菌ゲノムの領域に挿入されます この領域が転写されると 生成物はtracrRNAと呼ばれる 小さなRNAと結合し Cas9タンパク質とRNAseの両方を 分子に向けるのに役立ち 後者は転写産物を切断します 最終的に幾つかの複合体となり それぞれ遺伝子座のDNAに由来する Cas9タンパク質(tracrRNA)およびCRISPR-RNAからなっています これらの構造のCRISPR-RNAは Cas9を認識してウイルスDNAに導き ウイルスDNAは次に 切断されて破壊されます 科学者たちは 関心のある遺伝子を標的とする tracrRNAおよびcrRNAを模倣する 個々のRNA分子を合成して CRISPR-Cas9を利用します 例えば そのようなガイドRNA2つが Cas9のある細胞に導入され 両方が同じ遺伝子を標的とする場合 配列が切除されます この標的領域が除去されると 切断端は再結合され 細胞への影響が観察されます このようにCRISPR-Cas9システムは 細菌のメカニズムから改良されて 数々の遺伝子編集技術に使用されます

「最先端の分子生物学:学部生のためのCRISPR/CAS9遺伝子編集のレビュー」 生化学および分子生物学教育 46、no. 2 (2018): 195–205.[<a href="https://doi.org/10.1002/bmb.21108" target="__blank">ソース</a>]

Lander, Eric S. "The Heroes of CRISPR." Cell 164, no. 1 (2016年1月14日): 18–28.[<a href="https://doi.org/10.1016/j.cell.2015.12.041" target="__blank">ソース</a>]

crispr

ゲノム編集技術は、ゲノム上の特定の場所にある遺伝物質を追加、削除、または再配置することで、生物のDNAを修正できる技術です。このような技術は、血友病や鎌状赤血球貧血などの遺伝性疾患の治療に利用できる可能性があります。遺伝性疾患の安全で効果的な治療法につながる可能性のあるDNA編集研究ツールとして、CRISPR-Cas9システムが広く普及しています。CRISPR-Cas9とは、「Clustered Regularly Interspaced Short Palindromic Repeats」と「CRISPR-associated protein 9」の略です。基本的なCRISPR-Cas9システムは、Cas9エンドヌクレアーゼと、Cas9をターゲットDNAに導く低分子RNAから構成されています。 
 
 <h4>起源</h4
 
 CRISPR配列は、最初に細菌で観察され、その後、古細菌でも確認されました。研究者たちは、CRISPR-Cas9システムが、侵入してきたウイルスに対する適応免疫防御の役割を果たしていることを発見しました。多くの細菌とほとんどの古細菌は、ウイルスのDNAの短い配列を捕獲して、ウイルスのDNAセグメントのライブラリ、すなわちCRISPRアレイを作成します。原核生物が同じウイルスや同じクラスのウイルスに再びさらされると、CRISPRアレイは、ウイルスの侵入者を認識するのに役立つ小さなRNAセグメントを転写するのに使用され、その後、Cas9または同様のエンドヌクレアーゼでウイルスDNAを破壊します。 
 
 <h4>CRISPR-Cas9テクノロジーの利用</h4>
 
 CRISPR-Cas9は、DNAを除去して新しいDNA配列を挿入するために実験室でよく使われます。これを実現するために、研究者はまず、ゲノムDNA上の特定の標的配列に結合するガイド配列と呼ばれる短い配列を持つ、ガイドRNAと呼ばれる小さなRNAの断片を作成しなければなりません。また、ガイドRNAは、Cas9（またはCpf1などの他のエンドヌクレアーゼ）と結合できます。ガイドRNAとCas9タンパク質を目的の細胞に投与すると、ガイドRNAが標的DNA配列を特定し、Cas9がそれを切断します。 
 
 その後、細胞の機械がランダムなヌクレオチドを挿入または削除することで切断された鎖を修復し、標的遺伝子を不活性化します。また、ガイドRNAやCas9と一緒にカスタマイズしたDNA配列を細胞内に導入し、それを修復機構のテンプレートとして使用して、切り取られた配列を置き換えることもできます。この方法は、研究者が遺伝子をノックアウトしてその影響を調べたり、変異した遺伝子を正常なものに置き換えて病気を治したりするのに非常に効果的な方法です。 
 
 CRISPR-Cas9システムには大きな遺伝子改変能力があるため、その使用については、特に胚の編集に関して大きな議論があります。最近、中国の科学者が、CRISPR技術を使ってHIV感染に関わる遺伝子を無効化し、ゲノム編集された赤ちゃんを作ったと主張しました。これを受けて、倫理面や安全面での配慮を懸念する科学者たちが世界中で反発しました。多くの科学者がこの動きを時期尚早だとし、また他の科学者からはターゲット外のゲノムへの影響を懸念する声が上がっています。CRISPR-Cas9システムのバイオテクノロジーへの応用の可能性は数多くありますが、その使用によって生じる可能性のある将来的な課題を考慮することが重要です。

ゲノム編集技術により、科学者は特定の遺伝物質の追加、除去、または再配置を通じて生物のDNAを変更することができます...

Education

JoVE Core

生物学

Genetics

遺伝学

Biotechnology

バイオテクノロジー

KEY TERMS AND CONCEPTS

主要用語と概念

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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recombinant dna technology

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PCR Cycle for DNA Amplification

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complementary dna

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antibiotic selection

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in-vitro mutagenesis (gene knockouts)

Knockout Mice and Gene Replacement

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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

Gene Delivery for Serious Diseases

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embryonic stem cells

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adult stem cells

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induced pluripotent stem cells

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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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JoVE Core シリーズは、生物学の重要な概念を説明する 300 を超える簡潔でわかりやすいアニメーション ビデオ レッスンに加え、今日の研究室で行われる実際の研究実験を示す 150 以上の科学者の活動ビデオを通じて、生物学に命を吹き込みます。

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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.

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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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