Cas9中的突变提高了CRISPR-Cas免疫反应过程中病毒间隔序列的获取速率。
Mutations in Cas9 Enhance the Rate of Acquisition of Viral Spacer Sequences during the CRISPR-Cas Immune Response.
作者信息
Heler Robert, Wright Addison V, Vucelja Marija, Bikard David, Doudna Jennifer A, Marraffini Luciano A
机构信息
Laboratory of Bacteriology, The Rockefeller University, New York, NY 10065, USA.
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
出版信息
Mol Cell. 2017 Jan 5;65(1):168-175. doi: 10.1016/j.molcel.2016.11.031. Epub 2016 Dec 22.
CRISPR loci and their associated (Cas) proteins encode a prokaryotic immune system that protects against viruses and plasmids. Upon infection, a low fraction of cells acquire short DNA sequences from the invader. These sequences (spacers) are integrated in between the repeats of the CRISPR locus and immunize the host against the matching invader. Spacers specify the targets of the CRISPR immune response through transcription into short RNA guides that direct Cas nucleases to the invading DNA molecules. Here we performed random mutagenesis of the RNA-guided Cas9 nuclease to look for variants that provide enhanced immunity against viral infection. We identified a mutation, I473F, that increases the rate of spacer acquisition by more than two orders of magnitude. Our results highlight the role of Cas9 during CRISPR immunization and provide a useful tool to study this rare process and develop it as a biotechnological application.
CRISPR基因座及其相关（Cas）蛋白编码一种原核免疫系统，可抵御病毒和质粒。感染后，一小部分细胞会从入侵者那里获取短DNA序列。这些序列（间隔序列）被整合到CRISPR基因座的重复序列之间，使宿主对匹配的入侵者产生免疫。间隔序列通过转录成短RNA向导来指定CRISPR免疫反应的靶标，这些向导将Cas核酸酶导向入侵的DNA分子。在这里，我们对RNA引导的Cas9核酸酶进行了随机诱变，以寻找对病毒感染具有增强免疫力的变体。我们鉴定出一个突变，即I473F，它使间隔序列获取率提高了两个多数量级。我们的结果突出了Cas9在CRISPR免疫过程中的作用，并提供了一个有用的工具来研究这一罕见过程，并将其开发为一种生物技术应用。
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