Target preference of Type III-A CRISPR-Cas complexes at the transcription bubble

Area of Science:

• Microbiology
• Molecular Biology
• Immunology

Background:

• Type III-A CRISPR-Cas systems provide prokaryotic adaptive immunity against foreign DNA using Csm complexes.
• Csm complexes are known to cleave both RNA and single-stranded DNA (ssDNA).
• The specific target of Csm during transcription elongation remained unclear.

Purpose of the Study:

• To determine whether Type III-A CRISPR-Cas Csm complexes target RNA or ssDNA during transcription elongation.
• To investigate the interaction between Csm and the transcription elongation complex (TEC).

Main Methods:

• Biochemical experiments were conducted using Thermus thermophilus Csm (TthCsm) and Staphylococcus epidermidis Csm (SepCsm).
• The study analyzed the binding and cleavage activities of Csm within transcription elongation complexes (TECs).

Main Results:

• TthCsm binding to nascent transcripts in TECs promotes tethering to RNA polymerase (RNAP) without direct contact.
• Both TthCsm and SepCsm were shown to cleave RNA transcripts within the transcription bubble.
• Neither Csm complex demonstrated cleavage activity against ssDNA in the transcription bubble.

Conclusions:

• Type III-A CRISPR-Cas systems primarily target RNA transcripts for immunity against foreign DNA, rather than unwound ssDNA in TECs.
• This RNA-targeting mechanism in prokaryotes shares similarities with eukaryotic RNA interference pathways.
• The findings clarify the molecular mechanism of transcription-dependent immunity mediated by Csm complexes.