Genome editing / knockout 벡터 확립 및 도입

문서 내 토픽

• 1. Genome Editing

  Genome Editing은 생명체의 특정 염기서열을 교정하거나 편집하는 기술을 의미한다. 이러한 Genome editing 기술에는 과거부터 이용되었던 Zinc finger nucleases나 TALEN을 이용하는 방법이 있었으나 현재는 높은 정확도와 효율성을 갖추고, 비교적 쉽게 디자인할 수 있는 기술인 CRISPR을 주된 tool로써 사용하고 있다. CRISPR/Cas9 기술은 효소가 절단하는 부분의 염기서열을 guide RNA(gRNA)로서 인위적으로 제공해주어 원하는 부분의 DNA를 절단하도록 한다. 이를 이용해 특정 gene을 knock-in을 시키는 작업을 수행할 수 있다.

• 2. Genome Editing 벡터 제작

  Genome Editing 기술을 이용해서 삽입하고자 하는 double stranded guide DNA를 먼저 제작해야 한다. gDNA를 합성하기 위해서는 원하는 염기서열로 이루어진 두 종류의 primer를 상보적으로 합성하는 과정을 통해서 이루어진다. 이렇게 만들어진 guide DNA를 Cas9이 들어있는 Px459 벡터에 삽입하는 과정을 거친다. 제한효소와 Ligase를 이용하여 벡터에 guide DNA를 도입하고, 이를 세포에 transfection하는 과정을 거친다.

• 3. Transfection 방법

  Transfection은 외부 유전자를 다른 세포 내로 도입을 하는 과정으로, 바이러스를 이용하는 viral methods와 바이러스를 이용하지 않는 non-viral methods로 이루어져 있다. non-viral methods에는 physical methods와 chemical methods로 나뉘며, 방법은 비용, 시간, cell type 등을 고려해서 정해지게 된다. 대표적인 방법으로는 Calcium phosphate transfection, DEAE-dextran transfection, Cationic Lipids 를 사용하는 방법, Electroporation 등이 있다.

• 4. Prime Editing

  Prime Editing 기술은 기존 CRISPR/Cas9과 마찬가지로 특이적인 DNA sequence를 인식하지만 double-stranded DNA breaks가 아닌 single-stranded DNA breaks 방법을 사용한다. Prime Editing 기술에 필요한 component는 prime editing guide RNA(pegRNA)과 Cas9 그리고 reverse transcriptase이다. Cas9과 reverse transcriptase는 붙어서 fusion protein을 이룬다. pegRNA의 guide sequence의 도움을 받아 Cas9은 DNA의 한 가닥을 절단하고, reverse transcriptase가 절단된 부분의 DNA를 복구하게 된다. 이를 통해 더 정확한 genome editing이 가능하다.

Easy AI와 토픽 톺아보기

• 1. Genome Editing

  Genome editing is a powerful technology that allows for the precise modification of DNA sequences within living organisms. This technology has the potential to revolutionize various fields, including medicine, agriculture, and environmental science. By enabling the targeted alteration of genes, genome editing can be used to treat genetic disorders, enhance crop traits, and even address global challenges such as climate change. However, the ethical implications of this technology must be carefully considered, as it raises concerns about the potential for misuse and unintended consequences. Rigorous regulation and oversight are necessary to ensure that genome editing is used responsibly and for the benefit of humanity and the planet. As with any transformative technology, it is crucial to strike a balance between the immense potential of genome editing and the need to mitigate its risks.

• 2. Transfection Methods

  Transfection methods are the techniques used to introduce genetic material, such as DNA or RNA, into cells. These methods are essential for various applications in biotechnology, including genome editing, gene expression studies, and the production of recombinant proteins. The choice of transfection method depends on factors such as the cell type, the size and nature of the genetic cargo, and the desired efficiency of delivery. Common transfection methods include lipid-based transfection, electroporation, and viral transduction. Each method has its own advantages and limitations, and the selection of the appropriate technique is crucial for the success of the experiment or application. Ongoing research is focused on developing more efficient, targeted, and less cytotoxic transfection methods to expand the capabilities of genetic engineering and cell-based therapies. As the field of transfection continues to evolve, the availability of diverse and optimized transfection tools will be instrumental in unlocking the full potential of genome editing and other transformative biotechnologies.

• 3. Prime Editing

  Prime editing is a recently developed genome editing technology that offers a more precise and versatile approach compared to traditional CRISPR-Cas9 systems. Unlike the standard CRISPR method, which relies on the creation of double-strand breaks in the DNA, prime editing uses a modified CRISPR-Cas9 enzyme and a prime editing guide RNA (pegRNA) to directly convert one DNA base to another without the need for double-strand breaks. This approach allows for the introduction of a wider range of genetic modifications, including insertions, deletions, and base substitutions, with increased specificity and reduced off-target effects. Prime editing has the potential to address the limitations of previous genome editing tools, particularly in the context of correcting disease-causing mutations and engineering complex genetic changes. As this technology continues to be refined and optimized, it could significantly expand the applications of genome editing in both research and clinical settings, leading to advancements in areas such as gene therapy, disease modeling, and agricultural biotechnology. The development of prime editing represents an exciting milestone in the ongoing evolution of genome editing tools, and its continued progress will be crucial for unlocking new frontiers in the field of genetic engineering.