在下一代测序芯片上对CRISPR-Cas复合体进行大规模并行生物物理分析
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
概括
我们开发了一种名为CHAMP的新方法, 用以绘制数百万个DNA序列的CRISPR-Cas蛋白相互作用. 这揭示了CRISPR-Cas复合体如何与非目标DNA结合,改善了我们对基因编辑特异性的理解.
科学领域:
- 分子生物学
- 遗传学
- 生物化学
背景情况:
- CRISPR-Cas系统使用crRNA来准外来DNA.
- 对CRISPR-Cas目标外结合和核酶激活的定量理解有限.
研究的目的:
- 开发一种分析CRISPR-Cas蛋白与DNA相互作用的高通量方法.
- 通过I-ECRISPR-Cas (级联) 复合体和Cas3核酶进行DNA识别的全面调查.
主要方法:
- 重新使用下一代测序芯片来创建芯片混合协会映射平台 (CHAMP).
- 测量了蛋白质和大约10^7个独特DNA序列之间的同时相互作用.
- 分析了突变的目标序列和人类基因组DNA.
主要成果:
- 布识别了一个扩展的原空间器相邻动机 (PAM).
- 在DNA识别中表现出3核酸周期性.
- 通过PAM标识和PAM近位核酸,涉及Cse1子单元的释放来调节Cas3的招募.
结论:
- 开发了一种控制非目标DNA结合的生物物理约束模型.
- 通过CHAMP,可以对蛋白质-DNA相互作用进行高吞吐量定量分析.
- 这些发现有助于更好地理解CRISPR- Cas的特异性和潜在的非目标效应.
相关概念视频
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...
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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.
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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...
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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...


