title: Fragmentation of the CRISPR-Cas Type I-B signature protein Cas8b.
abstract: CRISPR arrays are transcribed into long precursor RNA species, which are further processed into mature CRISPR RNAs (crRNAs). Cas proteins utilize these crRNAs, which contain spacer sequences that can be derived from mobile genetic elements, to mediate immunity during a reoccurring virus infection. Type I CRISPR-Cas systems are defined by the presence of different Cascade interference complexes containing large and small subunits that play major roles during target DNA selection.
  
  
  
  Here, we produce the protein and crRNA components of the Type I-B CRISPR-Cas complex of Clostridium thermocellum and Methanococcus maripaludis. The C. thermocellum Cascade complexes were reconstituted and analyzed via size-exclusion chromatography. Activity of the heterologous M. maripaludis CRISPR-Cas system was followed using phage lambda plaques assays.
  
  
  
  The reconstituted Type-I-B Cascade complex contains Cas7, Cas5, Cas6b and the large subunit Cas8b. Cas6b can be omitted from the reconstitution protocol. The large subunit Cas8b was found to be represented by two tightly associated protein fragments and a small C-terminal Cas8b segment was identified in recombinant complexes and C. thermocellum cell lysate.
  
  
  
  Production of Cas8b generates a small C-terminal fragment, which is suggested to fulfill the role of the missing small subunit. A heterologous, synthetic M. maripaludis Type I-B system is active in E. coli against phage lambda, highlighting a potential for genome editing using endogenous Type-I-B CRISPR-Cas machineries. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.
authors.affiliation: Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch Strasse 10, 35043 Marburg, Germany.
authors.forename: Hagen
authors.initials: H
authors.lastname: Richter
authors.affiliation: Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch Strasse 10, 35043 Marburg, Germany.
authors.forename: Judith
authors.initials: J
authors.lastname: Rompf
authors.affiliation: Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch Strasse 10, 35043 Marburg, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Wiegel
authors.affiliation: Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch Strasse 10, 35043 Marburg, Germany.
authors.forename: Kristina
authors.initials: K
authors.lastname: Rau
authors.affiliation: Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch Strasse 10, 35043 Marburg, Germany. Electronic address: lennart.randau@mpi-marburg.mpg.de.
authors.forename: Lennart
authors.initials: L
authors.lastname: Randau
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D064130:CRISPR-Associated Proteins / Q000235:genetics* / Q000378:metabolism; D064113:CRISPR-Cas Systems / Q000235:genetics*; D003001:Cloning, Molecular; D048013:Acetivibrio thermocellus / Q000235:genetics / Q000378:metabolism; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D017017:Methanococcus / Q000235:genetics; D012323:RNA Processing, Post-Transcriptional; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics
chemical_list: D019843:Archaeal Proteins; D001426:Bacterial Proteins; D064130:CRISPR-Associated Proteins; D011993:Recombinant Fusion Proteins
keywords: CRISPR-Cas; Cascade; DNA interference; Ribonucleoproteins