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関連する概念動画

CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

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The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
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CRISPR01:59

CRISPR

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Genome editing technologies allow scientists to modify an organism’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...
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Homologous Recombination02:31

Homologous Recombination

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The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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CRISPR and crRNAs02:53

CRISPR and crRNAs

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Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...
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Improving Translational Accuracy02:07

Improving Translational Accuracy

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Base complementarity between the three base pairs of mRNA codon and the tRNA anticodon is not a failsafe mechanism. Inaccuracies can range from a single mismatch to no correct base pairing at all. The free energy difference between the correct and nearly correct base pairs can be as small as 3 kcal/ mol. With complementarity being the only proofreading step, the estimated error frequency would be one wrong amino acid in every 100 amino acids incorporated. However, error frequencies observed in...
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Proofreading01:31

Proofreading

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Synthesis of new DNA molecules is carried out by the enzyme DNA polymerase, which adds nucleotides on the daughter strand complementary to the template DNA strand. DNA polymerase has a higher affinity to add the correct base and ensures fidelity during DNA replication. Furthermore,  it exhibits proofreading activity during replication, using an exonuclease domain that cuts off incorrect nucleotides from the nascent DNA strand.
Errors During Replication are Corrected by the DNA Polymerase...
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CIRCLE-Seq for Interrogation of Off-Target Gene Editing
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改良された校正は,CRISPR-Cas9のターゲティングの精度を左右する.

Janice S Chen1, Yavuz S Dagdas2, Benjamin P Kleinstiver3,4,5

  • 1Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.

Nature
|September 21, 2017
PubMed
まとめ

新しいCRISPR- Cas9変種 (SpCas9- HF1とeSpCas9 ((1. 1)) は,標的外効果の減少を示しています. 新しい超正確な変種 (HypaCas9) が開発され,標的の活性を失うことなくゲノム編集の特異性を改善しました.

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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

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科学分野:

  • 分子生物学
  • 遺伝学
  • 生物化学

背景:

  • ストレプトコッカス・ピオゲネス (SpCas9) のCRISPR-Cas9システムは ゲノム編集の強力なツールです
  • 既存の高精度変種 (SpCas9-HF1,eSpCas9 ((1.1)) は,オフターゲットの割れ目を軽減しますが,それらのターゲットの差別化メカニズムは不明です.
  • 精密なゲノム工学のために,Cas9の特異性をさらに改善する必要があります.

研究 の 目的:

  • SpCas9の変種における標的差別の基礎となるメカニズムを解明する.
  • 新しいCas9変種を設計し 特殊性を高め 標的の活動を維持する
  • Cas9の標的認識と核酵素活性化のための洗練されたモデルを開発する.

主な方法:

  • SpCas9の変種を研究するために単分子フォースター共振エネルギー転送 (smFRET) 実験が採用されました.
  • 標的の結合と形状の変化を分析するために生体物理的手法を使用した.
  • 新しいCas9変種であるHypaCas9が設計され,全ゲノム特異性と標的活性について試験された.

主要な成果:

  • SpCas9-HF1とeSpCas9 ((1.1) は,不一致したDNA標的を結合するとき,不活性状態を採用する.
  • Cas9のREC3ドメインは,標的互補性を認識し,ヌクレアース活性を制御する上で重要な役割を果たします.
  • 新しく設計されたHypaCas9変種は,ヒト細胞における優越した全ゲノム特異性と堅固なオンターゲット編集を示しています.

結論:

  • REC3ドメインは,標的DNAのマッチングに基づいて,Cas9の触媒能力の重要なレギュラーである.
  • このメカニズムを理解することで より正確な CRISPR-Cas9 ツールの合理的な設計が可能になります
  • HypaCas9は 精密ゲノム編集技術の 重要な進歩を表しています