In vitro assembly and activity of an archaeal CRISPR-Cas type I-A Cascade interference complex.
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.
Prokaryotic Small RNA Biology Group, Max Planck Institute for Terrestrial Microbiology, D-35043 Marburg, Germany, Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany, Cell Biology and LOEWE Research Centre for Synthetic Microbiology, Philipps-Universität Marburg, D-35043 Marburg, Germany and Department of Structural Cell Biology, Max Planck Institute of Biochemistry, D-82152 Martinsried, Germany.
André
A
Plagens
Vanessa
V
Tripp
Michael
M
Daume
Kundan
K
Sharma
Andreas
A
Klingl
Ajla
A
Hrle
Elena
E
Conti
Henning
H
Urlaub
Lennart
L
Randau
D019843:Archaeal Proteins / Q000378:metabolism*; D064130:CRISPR-Associated Proteins / Q000378:metabolism*; D064113:CRISPR-Cas Systems; D053837:DNA Cleavage; D019641:DNA, Archaeal / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D019642:RNA, Archaeal / Q000737:chemistry / Q000378:metabolism; D041542:Thermoproteus / Q000201:enzymology / Q000235:genetics
D019843:Archaeal Proteins; D064130:CRISPR-Associated Proteins; D019641:DNA, Archaeal; D004277:DNA, Single-Stranded; D019642:RNA, Archaeal; D003851:Deoxyribonucleases; D005090:Exodeoxyribonucleases