Characterization of CRISPR RNA processing in Clostridium thermocellum and Methanococcus maripaludis

Abstract

The CRISPR arrays found in many bacteria and most archaea are transcribed into a long precursor RNA that is processed into small clustered regularly interspaced short palindromic repeats (CRISPR) RNAs (crRNAs). These RNA molecules can contain fragments of viral genomes and mediate, together with a set of CRISPR-associated (Cas) proteins, the prokaryotic immunity against viral attacks. CRISPR/Cas systems are diverse and the Cas6 enzymes that process crRNAs vary between different subtypes. We analysed CRISPR/Cas subtype I-B and present the identification of novel Cas6 enzymes from the bacterial and archaeal model organisms Clostridium thermocellum and Methanococcus maripaludis C5. Methanococcus maripaludis Cas6b in vitro activity and specificity was determined. Two complementary catalytic histidine residues were identified. RNA-Seq analyses revealed in vivo crRNA processing sites, crRNA abundance and orientation of CRISPR transcription within these two organisms. Individual spacer sequences were identified with strong effects on transcription and processing patterns of a CRISPR cluster. These effects will need to be considered for the application of CRISPR clusters that are designed to produce synthetic crRNAs.

Structure and RNA-binding properties of the Type III-A CRISPR-associated protein Csm3

“…The crRNA (locus 5, spacer 5) was produced by in vitro run-off transcription and purified by elution of the crRNA transcript from a polyacrylamide gel as described. 10 The RNA molecules were 5'-labeled with T4 polynucleotide kinase (New England Biolabs) and γ-[…”

“…5 Upon infection, these clusters are transcribed into precursor crRNAs (pre-crRNA), which then are processed into mature CRISPR RNAs (crRNA). [8][9][10] The common features of mature crRNAs are the spacer, which identifies the matching target (protospacer) via base pairing, and the 5'-terminal 8 nt repeat tag (psi-tag), which is complementary to the self DNA but not to 2-4 nt short protospacer adjacent motif (PAM) sequences. 11 Adjacent to this array are the cas genes.…”

CRISPR-mediated defense mechanisms in the hyperthermophilic archaeal genusSulfolobus

“…52 The presence of bidirectional transcripts has been found in few archaeal species: Sulfolobus spp, P. furiosus and Methanococcus maripaludis. 13,70 In bacteria, with the exception of Clostridium thermocellum, no antisense transcripts were detected and CRISPR transcription seems to be mostly classification merged the different CRISPR families into three major types: type I, type II, type III, which can be further subdivided. 46 Several CRISPR types can be present simultaneously on the chromosome of the same organism, indicating that each type could have different activities against various genetic elements or nucleic acids.…”

“…28 The process was shown to be dependent on a small recognition motif called PAM (protospacer adjacent motif) 10,12,47,75 located in the vicinity of the uni-directional. 11,39,40,70 Although several hypotheses have been formulated, the biological importance of bidirectional transcription remains unclear and no experimental data have so far elucidated the role of antisense RNAs in CRISPR regulation or interference. 55,71 The presence of several putative regulatory motifs within the leader sequence, and the high energetic cost related to the possible constitutive expression of a long non-coding RNA (precrRNA) in the absence of an invading genetic element, speaks for a complex transcriptional regulation of the CRISPR locus.…”

Characterization of CRISPR RNA transcription by exploiting stranded metatranscriptomic data

“…Antisense RNAs of crRNAs were detected in a few species, including Clostridium thermocellum (Richter et al 2012), Sulfolobu acidocaldarius (Lillestol et al 2009), and Pyrococcus furiosus (Juranek et al 2012). In general, the abundance of antisense crRNAs is lower than their crRNA counterparts.…”