CRISPR-Cas systems provide adaptive immunity in bacteria and archaea, beginning with integration of foreign sequences into the host CRISPR genomic locus and followed by transcription and maturation of CRISPR RNAs (crRNAs). In some CRISPR systems, a reverse transcriptase (RT) fusion to the Cas1 integrase and Cas6 maturase creates a single protein that enables concerted sequence integration and crRNA production. To elucidate how the RT-integrase organizes distinct enzymatic activities, we present the cryo-EM structure of a Cas6-RT-Cas1—Cas2 CRISPR integrase complex. The structure reveals a heterohexamer in which the RT directly contacts the integrase and maturase domains, suggesting functional coordination between all three active sites. Together with biochemical experiments, our data support a model of sequential enzymatic activities that enable CRISPR sequence acquisition from RNA and DNA substrates. These findings highlight an expanded capacity of some CRISPR systems to acquire diverse sequences that direct CRISPR-mediated interference.

CRISPR-Cas 系统在细菌和古细菌中提供适应性免疫，首先将外源序列整合到宿主 CRISPR 基因组位点，然后是 CRISPR RNA (crRNA) 的转录和成熟。在一些 CRISPR 系统中，逆转录酶 (RT) 与 Cas1 整合酶和 Cas6 成熟酶的融合产生了一种单一的蛋白质，可实现协同的序列整合和 crRNA 生产。为了阐明 RT 整合酶如何组织不同的酶活性，我们展示了 Cas6-RT-Cas1-Cas2 CRISPR 整合酶复合物的冷冻电镜结构。该结构揭示了异六聚体，其中 RT 直接接触整合酶和成熟酶域，表明所有三个活性位点之间存在功能协调。结合生化实验，我们的数据支持序列酶活性模型，该模型能够从 RNA 和 DNA 底物获取 CRISPR 序列。这些发现突出了一些 CRISPR 系统获得引导 CRISPR 介导干扰的不同序列的能力的扩展。