III-A 型 CRISPR-Cas 系统 Csm 复合物的遗传剖析揭示了各个亚基的作用。
Genetic Dissection of the Type III-A CRISPR-Cas System Csm Complex Reveals Roles of Individual Subunits.
机构信息
Institute of Biotechnology, Vilnius University, Saulėtekio av. 7, 10257 Vilnius, Lithuania.
Institute of Biotechnology, Vilnius University, Saulėtekio av. 7, 10257 Vilnius, Lithuania.
出版信息
Cell Rep. 2019 Mar 5;26(10):2753-2765.e4. doi: 10.1016/j.celrep.2019.02.029.
The type III-A Csm complex of Streptococcus thermophilus (StCsm) provides immunity against invading nucleic acids through the coordinated action of three catalytic domains: RNase (Csm3), ssDNase (Cas10-HD), and cyclic oligoadenylates synthase (Cas10-Palm). The matured StCsm complex is composed of Cas10:Csm2:Csm3:Csm4:Csm5 subunits and 40-nt CRISPR RNA (crRNA). We have carried out gene disruptions for each subunit and isolated deletion complexes to reveal the role of individual subunits in complex assembly and function. We show that the Cas10-Csm4 subcomplex binds the 5'-handle of crRNA and triggers Csm3 oligomerization to form a padlock for crRNA binding. We demonstrate that Csm5 plays a key role in target RNA binding while Csm2 ensures RNA cleavage at multiple sites by Csm3. Finally, guided by deletion analysis, we engineered a minimal Csm complex containing only the Csm3, Csm4, and Cas10 subunits and crRNA and demonstrated that it retains all three catalytic activities, thus paving the way for practical applications.
嗜热链球菌的 III-A 型 Csm 复合物（StCsm）通过三个催化结构域的协调作用提供对入侵核酸的免疫：核糖核酸酶（Csm3）、单链 DNA 酶（Cas10-HD）和环寡腺苷酸合酶（Cas10-Palm）。成熟的 StCsm 复合物由 Cas10：Csm2：Csm3：Csm4：Csm5 亚基和 40nt CRISPR RNA（crRNA）组成。我们对每个亚基进行了基因敲除，并分离了缺失复合物，以揭示各个亚基在复合物组装和功能中的作用。我们表明 Cas10-Csm4 亚复合物结合 crRNA 的 5'-柄，并触发 Csm3 寡聚化形成 crRNA 结合的锁扣。我们证明 Csm5 在靶 RNA 结合中起着关键作用，而 Csm2 通过 Csm3 确保在多个位点切割 RNA。最后，在缺失分析的指导下，我们设计了一个仅包含 Csm3、Csm4 和 Cas10 亚基和 crRNA 的最小 Csm 复合物，并证明它保留了所有三种催化活性，从而为实际应用铺平了道路。
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