CRISPR-Cas9 环形变构体作为基因组修饰的可编程支架。
CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification.
机构信息
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Gladstone Institutes, San Francisco, CA 94158, USA.
出版信息
Cell. 2019 Jan 10;176(1-2):254-267.e16. doi: 10.1016/j.cell.2018.11.052.
The ability to engineer natural proteins is pivotal to a future, pragmatic biology. CRISPR proteins have revolutionized genome modification, yet the CRISPR-Cas9 scaffold is not ideal for fusions or activation by cellular triggers. Here, we show that a topological rearrangement of Cas9 using circular permutation provides an advanced platform for RNA-guided genome modification and protection. Through systematic interrogation, we find that protein termini can be positioned adjacent to bound DNA, offering a straightforward mechanism for strategically fusing functional domains. Additionally, circular permutation enabled protease-sensing Cas9s (ProCas9s), a unique class of single-molecule effectors possessing programmable inputs and outputs. ProCas9s can sense a wide range of proteases, and we demonstrate that ProCas9 can orchestrate a cellular response to pathogen-associated protease activity. Together, these results provide a toolkit of safer and more efficient genome-modifying enzymes and molecular recorders for the advancement of precision genome engineering in research, agriculture, and biomedicine.
工程天然蛋白质的能力对未来的实用生物学至关重要。CRISPR 蛋白已经彻底改变了基因组修饰，但 CRISPR-Cas9 支架不适合融合或细胞触发的激活。在这里，我们表明，使用环状排列对 Cas9 进行拓扑重排为 RNA 引导的基因组修饰和保护提供了一个先进的平台。通过系统询问，我们发现蛋白质末端可以定位在结合的 DNA 旁边，为战略性融合功能域提供了一种简单的机制。此外，环状排列使蛋白酶感应 Cas9（ProCas9）成为可能，ProCas9 是一类独特的单分子效应物，具有可编程的输入和输出。ProCas9 可以感应广泛的蛋白酶，我们证明 ProCas9 可以协调细胞对病原体相关蛋白酶活性的反应。总之，这些结果为研究、农业和生物医学中精确基因组工程的发展提供了更安全、更高效的基因组修饰酶和分子记录器工具包。
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