Biochemical characterization of RNA-guided ribonuclease activities for CRISPR-Cas9 systems.
Biochemical characterization of RNA-guided ribonuclease activities for CRISPR-Cas9 systems.
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DOI：
10.1016/j.ymeth.2019.06.018
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发表时间：
2020-02
期刊：
影响因子：
4.8
通讯作者：
Max J Gramelspacher;Zhonggang Hou;Yan Zhang
中科院分区：
文献类型：
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作者：
Max J Gramelspacher;Zhonggang Hou;Yan Zhang
大多数细菌和古细菌依赖于CRISPR-Cas系统来针对移动的遗传元件进行RNA引导的适应性免疫。II型CRISPR相关DNA内切核酸酶的Cas9家族在侧接PAM基序的CRISPR互补DNA靶标中产生可编程的双链断裂。如今，CRISPR-Cas9为真核生物和原核生物的精确基因组操作提供了一套强大的工具。最近，一些Cas9直向同源物被报道具有内在的CRISPR引导的序列特异性核糖核酸酶活性。这些发现从根本上扩展了CRISPR-Cas9系统的靶向能力，并有望提供新的CRISPR工具来操纵特定的细胞RNA转录物。在这里，我们提出了一个详细的方法，用于生化表征Cas9的RNA靶向潜力。
The majority of bacteria and archaea rely on CRISPR-Cas systems for RNA-guided, adaptive immunity against mobile genetic elements. The Cas9 family of type II CRISPR-associated DNA endonucleases generates programmable double strand breaks in the CRISPR-complementary DNA targets flanked by the PAM motif. Nowadays, CRISPR-Cas9 provides a set of powerful tools for precise genome manipulation in eukaryotes and prokaryotes. Recently, a few Cas9 orthologs have been reported to possess intrinsic CRISPR-guided, sequence-specific ribonuclease activities. These discoveries fundamentally expanded the targeting capability of CRISPR-Cas9 systems, and promise to provide new CRISPR tools to manipulate specific cellular RNA transcripts. Here we present a detailed method for the biochemical characterization of Cas9′s RNA-targeting potential.