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学術論文

In vitro assembly and activity of an archaeal CRISPR-Cas type I-A Cascade interference complex.

MPS-Authors
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Sharma,  K.
Research Group of Bioanalytical Mass Spectrometry, MPI for Biophysical Chemistry, Max Planck Society;

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Urlaub,  H.
Research Group of Bioanalytical Mass Spectrometry, MPI for Biophysical Chemistry, Max Planck Society;

フルテキスト (公開)

2032469.pdf
(出版社版), 5MB

付随資料 (公開)

2032469_Suppl.pdf
(付録資料), 693KB

引用

Plagens, A., Tripp, V., Daume, M., Sharma, K., Klingl, A., Hrle, A., Conti, E., Urlaub, H., & Randau, L. (2014). In vitro assembly and activity of an archaeal CRISPR-Cas type I-A Cascade interference complex. Nucleic Acids Research, 42(8), 5125-5138. doi:10.1093/nar/gku120.


引用: http://hdl.handle.net/11858/00-001M-0000-0019-B655-4
要旨
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) systems of type I use a Cas ribonucleoprotein complex for antiviral defense (Cascade) to mediate the targeting and degradation of foreign DNA. To address molecular features of the archaeal type I-A Cascade interference mechanism, we established the in vitro assembly of the Thermoproteus tenax Cascade from six recombinant Cas proteins, synthetic CRISPR RNAs (crRNAs) and target DNA fragments. RNA-Seq analyses revealed the processing pattern of crRNAs from seven T. tenax CRISPR arrays. Synthetic crRNA transcripts were matured by hammerhead ribozyme cleavage. The assembly of type I-A Cascade indicates that Cas3′ and Cas3′′ are an integral part of the complex, and the interference activity was shown to be dependent on the crRNA and the matching target DNA. The reconstituted Cascade was used to identify sequence motifs that are required for efficient DNA degradation and to investigate the role of the subunits Cas7 and Cas3′′ in the interplay with other Cascade subunits.