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キーワード:
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要旨:
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR associated) systems are present in many prokaryotes and provide adaptive immunity against viruses and other mobile genetic elements. They can recognize new foreign genetic material through adaptation, which involves the integration of short segments of foreign DNA into an extending CRISPR array. This process allows the system to evolve continually and to provide a robust defense against a wide range of invaders. Depending on the involved Cas proteins, CRISPR-Cas systems are classified into different types and two major classes. Type I systems use a ribonucleoprotein complex termed Cascade (CRISPR-associated complex for antiviral defense) to scan for invading DNA and bind to the sequence that matches its crRNA (CRISPR RNA). Once the target is identified, the nuclease Cas3 is recruited and degrades the DNA in a process known as interference. The Type IV CRISPR-Cas system is a member of Class 1 and has three subtypes. This study focuses specifically on the Type IV-A system, which is characterized by the absence of a DNA nuclease, the presence of a CRISPR array, the lack of an adaptation module, and an association with the helicase DinG. To examine the biological function of the Type IV-A CRISPR-Cas system, Pseudomonas oleovorans was selected as a model organism and found to contain a Type IV-A CRISPR-Cas system on its megaplasmid. Bioinformatic analyses of its CRISPR array showed that one of the crRNAs targets the gene pilN in the genome, providing evidence of adaptation of host sequences. A 5′-AAG-3′ PAM sequence was found to be shared between the Type IV-A and Type I-E CRISPR-Cas systems, suggesting the possibility of cross-talk at the level of adaptation. Deep sequencing of genomic DNA of P. oleovorans cells electroporated with pre-spacers revealed spacer rearrangements and the presence of new spacers. In vivo assays in P. oleovorans and E. coli recombinant systems demonstrated that the Type IV-A system conducts PAM-dependent interference. This system exhibits anti-plasmid activity by targeting an open reading frame within one of the multiple cloning sites. Furthermore, it displays resistance to lambda phage infection by targeting gene E, which encodes a crucial head protein. Observation of efficient lacZ reporter targeting provided the first evidence that the Type IV-A system conducts interference without DNA degradation. Therefore, the Type IV-A CRISPR-Cas system holds great promise as a powerful transcription modulation tool with a natural CRISPRi-like mechanism.
