Fragmentation of the CRISPR-Cas Type I-B signature protein Cas8b

• Published in: Biochimica et biophysica acta. General subjects Vol. 1861; no. 11; pp. 2993 - 3000
• Main Authors: Richter, Hagen, Rompf, Judith, Wiegel, Julia, Rau, Kristina, Randau, Lennart
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.11.2017
• Subjects: Amino Acid Sequence; Archaeal Proteins - genetics; Archaeal Proteins - metabolism; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; bacteriophages; Cascade; Cloning, Molecular; Clostridium thermocellum; Clostridium thermocellum - genetics; Clostridium thermocellum - metabolism; Clustered Regularly Interspaced Short Palindromic Repeats - genetics; CRISPR-Associated Proteins - genetics; CRISPR-Associated Proteins - metabolism; CRISPR-Cas; CRISPR-Cas Systems - genetics; DNA; DNA interference; Escherichia coli; gel chromatography; genome; immunity; interspersed repetitive sequences; Methanococcus - genetics; Methanococcus maripaludis; proteins; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Ribonucleoproteins; RNA precursors; RNA Processing, Post-Transcriptional; Signal Transduction - genetics; synthetic biology; CRISPR-Cas; Cascade; Ribonucleoproteins; DNA interference
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/j.bbagen.2017.02.026

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. •An archaeal Type I-B CRISPR-Cas system is active in E. coli against phage lambda.•Recombinant Type I-B Cascade was reconstituted in vivo.•Recombinant Cas8b production yields a small fragment in bacteria and archaea.•The small Cas8b fragment could compensate for the lack of a small Cascade subunit.