Programmable RNA recognition and cleavage by CRISPR/Cas9.
Programmable RNA recognition and cleavage by CRISPR/Cas9.
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DOI：
10.1038/nature13769
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发表时间：
2014-12-11
期刊：
影响因子：
64.8
通讯作者：
Doudna, Jennifer A.
中科院分区：
文献类型：
--
作者：
O'Connell, Mitchell R.;Oakes, Benjamin L.;Sternberg, Samuel H.;East-Seletsky, Alexandra;Kaplan, Matias;Doudna, Jennifer A.
CRISPR 相关蛋白 Cas9 是一种 RNA 引导的 DNA 核酸内切酶，它利用 RNA:DNA 互补性来识别序列特异性双链 DNA (dsDNA) 切割的靶位点。在其天然环境中，Cas9 专门作用于 DNA 底物，因为结合和催化都需要识别短 DNA 序列，即原型间隔子相邻基序 (PAM)，该序列位于 dsDNA 中与 20 核苷酸靶位点相对的链旁边和相对的链上。 Cas9 已被证明是多种细胞类型和生物体中基因组工程和基因调控的通用工具，但它被认为无法靶向 RNA。在这里，我们表明，当 PAM 作为单独的 DNA 寡核苷酸反式存在时，Cas9 以高亲和力与与 Cas9 相关指导 RNA 序列匹配的单链 RNA (ssRNA) 靶标结合。此外，PAM 呈递寡核苷酸 (PAMmers) 刺激 ssRNA 靶标的位点特异性核酸内切裂解，类似于 PAM 介导的 Cas9 催化 DNA 裂解的刺激。使用专门设计的 PAMmers，Cas9 可以专门结合或切割 RNA 靶标，同时避开相应的 DNA 序列，我们证明这种策略能够从细胞中分离特定的内源 mRNA。这些结果揭示了 PAM 结合与 Cas9 的底物选择之间的基本联系，并强调了 Cas9 在可编程和无标签转录物识别方面的实用性。
The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA:DNA complementarity to identify target sites for sequence-specific doublestranded DNA (dsDNA) cleavage. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, the protospacer adjacent motif (PAM), next to and on the strand opposite the 20-nucleotide target site in dsDNA. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in many cell types and organisms, but it has been thought to be incapable of targeting RNA. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalyzed DNA cleavage. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous mRNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable and tagless transcript recognition.
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