Jakhanwal Shrutee, Cress Brady F, Maguin Pascal, Lobba Marco J, Marraffini Luciano A, Doudna Jennifer A
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA.
Nucleic Acids Res. 2021 Apr 6;49(6):3546-3556. doi: 10.1093/nar/gkab123.
CRISPR-Cas9 is an RNA-guided DNA endonuclease involved in bacterial adaptive immunity and widely repurposed for genome editing in human cells, animals and plants. In bacteria, RNA molecules that guide Cas9's activity derive from foreign DNA fragments that are captured and integrated into the host CRISPR genomic locus by the Cas1-Cas2 CRISPR integrase. How cells generate the specific lengths of DNA required for integrase capture is a central unanswered question of type II-A CRISPR-based adaptive immunity. Here, we show that an integrase supercomplex comprising guide RNA and the proteins Cas1, Cas2, Csn2 and Cas9 generates precisely trimmed 30-base pair DNA molecules required for genome integration. The HNH active site of Cas9 catalyzes exonucleolytic DNA trimming by a mechanism that is independent of the guide RNA sequence. These results show that Cas9 possesses a distinct catalytic capacity for generating immunological memory in prokaryotes.
CRISPR-Cas9是一种RNA引导的DNA内切核酸酶，参与细菌的适应性免疫，并被广泛应用于人类细胞、动物和植物的基因组编辑。在细菌中，引导Cas9活性的RNA分子来源于外来DNA片段，这些片段被Cas1-Cas2 CRISPR整合酶捕获并整合到宿主CRISPR基因组位点中。细胞如何产生整合酶捕获所需的特定长度的DNA是基于II-A型CRISPR的适应性免疫中一个尚未解决的核心问题。在这里，我们表明，一种由引导RNA以及Cas1、Cas2、Csn2和Cas9蛋白组成的整合酶超复合物会产生基因组整合所需的精确修剪的30个碱基对的DNA分子。Cas9的HNH活性位点通过一种独立于引导RNA序列的机制催化核酸外切DNA修剪。这些结果表明，Cas9在原核生物中具有产生免疫记忆的独特催化能力。
