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

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Links Among Engineering, Technology, Science, and Society

BIO1.ETS2.3

CRISPR

- [Anlatan] CRISPR-Cas9 sistemi, demetlenmiş, düzenli aralıklı, kısa palindromik tekrarları ve CRISPR ilişkili protein 9'u temsil eden bir DNA düzenleme aracıdır.
 İlk olarak bakterilerde gözlemlenen CRISPR-Cas9, virüslere karşı bir savunma imkanıdır.
 Yabancı virüs DNA'sı bakteriye girerken, küçük küçük parçalara ayrılır, küçük parçalar da CRISPR yeri denen bakteri genomu bölgesine eklenebilirler.
Bölge kopya edilirken, ürün tracrRNAs denen daha küçük RNA'lar ile birleşir, bu RNA'lar Cas9 proteinin ve kopyayı ikiye bölen RNAse'nin moleküle yönelmesine yardımcı olabilir.
 Nihai sonuç birçok komplekstir, her biri bu yerdeki DNA'dan türetilen bir Cas9 proteini, tracrRNA ve bir CRISPR-RNA'dan oluşur.
 Bu yapılardaki CRISPR-RNA Cas9'u tanır ve virüs DNA'sına yönlendirir, ardından bölünür ve tahrip olur.
Bilim insanları, ilgi duyulan bir geni hedefleyebilen tracrRNA ve crRNA'ya benzeyen münferit RNA moleküllerini sentezleyerek CRISPR-Cas9'dan yararlanmaktadır.
 Mesela, bunun gibi 2 kılavuz RNA Cas9 ile hücrelere tanıtılınca ve ikisi de aynı geni hedefleyince, bir dizi kesilebilir.
 Bu hedef bölge çıkarılınca, kesim uçları tekrar bağlanır ve hücrelerin üzerindeki etkiler gözlemlenir.
Böylece, CRISPR-Cas9 sistemi bir bakteri mekanizmasından modifiye edilir ve bir dizi gen düzenleme tekniği için kullanılabilir.

Thurtle-Schmidt, Deborah M. ve Te-Wen Lo. "En Son Teknolojide Moleküler Biyoloji: Lisans Öğrencileri için CRISPR/CAS9 Gen Düzenlemesi Üzerine Bir İnceleme." Biyokimya ve Moleküler Biyoloji Eğitimi 46, no. 2 (2018): 195–205. [<a href="https://doi.org/10.1002/bmb.21108" target="__blank">Kaynak</a>]

Lander, Eric S. "CRISPR Kahramanları." Hücre 164, no. 1 (14 Ocak 2016): 18–28. [<a href="https://doi.org/10.1016/j.cell.2015.12.041" target="__blank">Kaynak</a>]

crispr

Genom Düzenleme Teknolojileri, bilim adamlarının bir organizmanın DNA'sını, genetik materyallerin belirli genomik noktalarda ilave, kaldırılması veya yeniden düzenlenmesi yoluyla değiştirmelerini sağlar. Bu tür teknikler, hemofili ve orak hücre anemisi gibi genetik bozuklukları iyileştirmek için potansiyel olarak kullanılabilir. Genetik bozukluklar için güvenli ve etkili tedavilere yol açabilecek popüler ve yaygın olarak kullanılan bir DNA düzenleme araştırması aracı CRISPR-CAS9 sistemidir. CRISPR-Cas9, kümelenmiş düzenli aralıklı kısa Palindromik tekrarlar ve CRISPR ile ilişkili protein 9 anlamına gelir. Temel bir CRISPR-Cas9 sistemi, bir cas9 endonükleaz ve Cas9'u hedef DNA'ya yönlendiren küçük bir RNA'dan oluşur.

<h4>Kökeni</h4>

CRISPR dizileri ilk olarak bakterilerde gözlendi ve daha sonra arkelerde tanımlandı. Araştırmacılar CRISPR-Cas9 sisteminin istilacı virüslere karşı adaptif bir bağışıklık savunmasına hizmet ettiğini keşfettiler. Birçok bakteri ve arke bir DNA segment kütüphanesi ya da CROSPR dizisi oluşturmak için kısa viral DNA dizileri yakalar. Prokaryotlar aynı virüse veya virüs sınıfına yeniden maruz kaldığında, CRISPR dizilerini kullanarak viral istilacıları tanımaya yardımcı olan küçük RNA segmentlerini üretir ve daha sonra Cas9 veya benzer bir ensonükleaz ile viral DNA'yı yok ederler.

<h4>CRISPR-Cas9 Teknolojisi Kullanımı</h4>

CRISPR-Cas9 genellikle genomdan bir DNA parçası kaldırmak ve yerine yeni bir DNA dizisi eklemek için laboratuvarda kullanılır. Bunu başarmak için, araştırmacıların öncelikle kılavuz RNA adı verilen küçük bir RNA parçası oluşturmaları gerekir ve bu kısa bir dizi ile genomik DNA'daki belirli bir hedef diziye bağlanan kılavuz dizisi olarak adlandırılır. Kılavuz RNA da Cas9 (veya Cpf1 gibi diğer endonükleaz) ile ilişkilendirilebilir. Kılavuz RNA ve Cas9 proteini, RNA kılavuzunun hedef DNA dizisini tanımladığı ve Cas9'un onu kestiği bir hücreye uygulanır.

Hücrenin mekanizmaları daha sonra rasgele nükleotitler ekleyerek veya silerek kırık iplikçikleri onarır, hedef geni inaktif hale getirir. Alternatif olarak, özelleştirilmiş bir DNA dizisi, onarım mekanizmaları için bir şablon görevi görecek şekilde ve çıkarılan sıranın yerini alan RNA ve Cas9 kılavuzuyla birlikte hücreye eklenebilir. Bu araştırmacılar için genlerin "nakavt" etkilerini incelemek için ya da bir hastalığı tedavi etmek için mutasyona uğramış bir gen yerine normal bir kopyasını koyduklarını oldukça efektif bir yoldur.

<h4>İnsanlarda Etik ve Fizibilite Konuları</h4>

CRISPR-Cas9 sisteminin önemli gen modifikasyon yeteneklerinin bir sonucu olarak, özellikle embriyo düzenlemesi için kullanımı konusunda büyük tartışmalar yaşanmıştır. Çinli bir bilim adamı son zamanlarda HIV enfeksiyonu ile ilgili bir geni devre dışı bırakmak için CRISPR teknolojisini kullanarak genom düzenlenmiş bebekler yarattığını iddia etti. Bu, prosedürün etik ve güvenlik konuları konusunda endişe duyan bilim adamlarının küresel bir tepkisine yol açtı. Pek çok kişi bu hareketi erken olarak nitelendirirlerken, diğerleri hedef dışı genomik etkilerle ilgili endişelerini dile getirdiler. CRISPR-Cas9 sistemi için olası biyoteknoloji uygulamalarının sayısı büyük olmakla birlikte, kullanımı sonucunda ortaya çıkabilecek zorlukları göz önünde bulundurmak önemlidir.

Genom düzenleme teknolojileri, bilim adamlarının bir organizmanın DNA'sını, genetik materyalin belirli bir şekilde eklenmesi, çıkarılması veya yeniden düzenlenmesi yoluyla değiştirmesine izin verir.

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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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JoVE Core serisi, biyolojinin ana konseptlerini açıklayan 300 adet kısa ve kolay anlaşılabilir olan video dersleriyle ve günümüz laboratuvarlarında yürütülen gerçek araştırmaları gösteren 150'den fazla \"bilim insanları iş başında\" videolarıyla biyolojiye hayat vermektedir.

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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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