次世代のシーケンシングチップ上のCRISPR-Cas複合体の大規模並列生体分析
Cheulhee Jung1, John A Hawkins2, Stephen K Jones1
1Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA.
Cell
|July 1, 2017
まとめ
私たちは新しい方法 CHAMP を開発し CRISPR-Cas タンパク質の相互作用を 何百万もの DNA 配列にマッピングしました これはCRISPR-Cas複合体が 標的外DNAに結合する方法を明らかにし 遺伝子編集の特異性に関する理解を深めています
科学分野:
- 分子生物学
- 遺伝学
- 生物化学
背景:
- CRISPR-Casシステムは 異なったDNAを標的として crRNAを使用します
- CRISPR- Casの標的外結合と核酵素活性化の定量的な理解は限られている.
研究 の 目的:
- CRISPR-Casタンパク質とDNAの相互作用を分析するための高通量メソッドを開発する.
- I-E型CRISPR-Cas (カスケード) 複合体とCas3核酵素によるDNA認識を包括的に調査する.
主な方法:
- 次の世代のシーケンシングチップを再利用して,チップハイブリッド化アソシエーションマッピングプラットフォーム (CHAMP) を作成します.
- タンパク質と約 10^7 個の DNA 配列の相互作用を測定した.
- 変異した標的配列とヒトゲノムDNAを分析した
主要な成果:
- カスケードでは,拡張したプロトスペーサー隣接モチーフ (PAM) を認識します.
- カスケードはDNA認識において3核酸周期性を示す.
- Cas3の募集は,Cse1サブユニット放出を含むPAM同一性およびPAM近接核酸によって制御される.
結論:
- 対象外DNA結合を制御する生体学的制約のモデルを開発した.
- CHAMPはタンパク質とDNAの相互作用を 定量的に分析できます
- この発見は,CRISPR- Casの特異性と潜在的な非標的効果の理解を深める.
関連する概念動画
CRISPR
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 Short...
CRISPR and crRNAs
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...
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...
Next-generation Sequencing
The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
Next-Generation Sequencing Methods
Although all next-generation methods use different technologies, they all share a set of standard features.
Next-Generation Sequencing Methods
Although all next-generation methods use different technologies, they all share a set of standard features.
CRISPR
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 Short...
CRISPR/Cas9 Genome Editing
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...


