Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR-Cas system.
Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR-Cas system.
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DOI：
10.1101/gad.272153.115
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发表时间：
2016-02-15
影响因子：
10.5
通讯作者：
Terns MP
中科院分区：
文献类型：
--
作者：
Elmore JR;Sheppard NF;Ramia N;Deighan T;Li H;Terns RM;Terns MP
在这里，Elmore等人研究了III-B型Cmr复合物如何发挥作用，该复合物切割CRISPR RNA（crRNA）识别的入侵RNA。研究结果表明，Cmr复合物是一种新型的DNA核酸酶，由含有crRNA靶序列和前间隔区邻近基序（rPAM）的入侵RNA激活。CRISPR-Cas系统消除细菌和古细菌中的核酸入侵者。III-B型Cmr系统的效应子复合物切割由复合物的CRISPR RNA（crRNA）识别的侵入RNA。在这里，我们表明，入侵RNA也激活Cmr复合物切割DNA。如对其他III型系统所观察到的，Cmr通过依赖于质粒内crRNA靶序列的转录的机制消除强烈火球菌中的质粒入侵者。值得注意的是，我们发现，目标RNA本身诱导DNA裂解的Cmr复合物在体外。DNA切割活性不依赖于靶RNA的切割，但特别需要在活化靶RNA内存在与靶序列相邻的短序列（rPAM [RNA前间隔区序列相邻基序]）。活化的复合物不需要DNA底物中的靶序列（或PAM）。通过P. furiosus Cmr系统消除质粒也不需要Csx 1（CRISPR相关Rossman fold [CARF]超家族）蛋白。质粒沉默依赖于Cmr 2（Cas 10超家族）蛋白的HD核酸酶和Palm结构域。结果确立了Cmr复合物作为由含有crRNA靶序列和rPAM的入侵RNA激活的新型DNA核酸酶。
Here, Elmore et al. investigate how the Type III-B Cmr complex, which cleaves invader RNAs recognized by the CRISPR RNA (crRNA), functions. The findings demonstrate that the Cmr complex is a novel DNA nuclease activated by invader RNAs containing a crRNA target sequence and a protospacer-adjacent motif (rPAM). CRISPR–Cas systems eliminate nucleic acid invaders in bacteria and archaea. The effector complex of the Type III-B Cmr system cleaves invader RNAs recognized by the CRISPR RNA (crRNA ) of the complex. Here we show that invader RNAs also activate the Cmr complex to cleave DNA. As has been observed for other Type III systems, Cmr eliminates plasmid invaders in Pyrococcus furiosus by a mechanism that depends on transcription of the crRNA target sequence within the plasmid. Notably, we found that the target RNA per se induces DNA cleavage by the Cmr complex in vitro. DNA cleavage activity does not depend on cleavage of the target RNA but notably does require the presence of a short sequence adjacent to the target sequence within the activating target RNA (rPAM [RNA protospacer-adjacent motif]). The activated complex does not require a target sequence (or a PAM) in the DNA substrate. Plasmid elimination by the P. furiosus Cmr system also does not require the Csx1 (CRISPR-associated Rossman fold [CARF] superfamily) protein. Plasmid silencing depends on the HD nuclease and Palm domains of the Cmr2 (Cas10 superfamily) protein. The results establish the Cmr complex as a novel DNA nuclease activated by invader RNAs containing a crRNA target sequence and a rPAM.