Adaptation by Type V-A and V-B CRISPR-Cas Systems Demonstrates Conserved Protospacer Selection Mechanisms Between Diverse CRISPR-Cas Types

Adaptation of clustered regularly interspaced short palindromic repeats (CRISPR) arrays is a crucial process responsible for the unique, adaptive nature of CRISPR-Cas immune systems. The acquisition of new CRISPR spacers from mobile genetic elements has previously been studied for several types of CRISPR-Cas systems. In this study, we used a...

Craspase is a CRISPR RNA-guided, RNA-activated protease

The CRISPR-Cas type III-E RNA-targeting effector complex gRAMP/Cas7-11 is associated with a caspase-like protein (TPR-CHAT/Csx29) to form Craspase (CRISPR-guided caspase). Here, we use cryo-electron microscopy snapshots of Craspase to explain its target RNA cleavage and protease activation mechanisms. Target-guide pairing extending into the 5...

The gRAMP CRISPR-Cas effector is an RNA endonuclease complexed with a caspase-like peptidase

Type III CRISPR-Cas immunity is widespread in prokaryotes and is generally mediated by multisubunit effector complexes. These complexes recognize complementary viral transcripts and can activate ancillary immune proteins. Here, we describe a type III-E effector from Candidatus “Scalindua brodae” (Sb-gRAMP), which is natively encoded by a...

Cas4-Cas1 Is a Protospacer Adjacent Motif-Processing Factor Mediating Half-Site Spacer Integration during CRISPR Adaptation

The immunization of bacteria and archaea against invading viruses via CRISPR adaptation is critically reliant on the efficient capture, accurate processing, and integration of CRISPR spacers into the host genome. The adaptation proteins Cas1 and Cas2 are sufficient for successful spacer acquisition in some CRISPR-Cas systems. However, many...

Mechanism for Cas4-assisted directional spacer acquisition in CRISPR–Cas

Prokaryotes adapt to challenges from mobile genetic elements by integrating spacers derived from foreign DNA in the CRISPR array¹. Spacer insertion is carried out by the Cas1–Cas2 integrase complex^2–4. A substantial fraction of CRISPR–Cas systems use a Fe–S cluster containing Cas4 nuclease to ensure that spacers are...

An educational guide for nanopore sequencing in the classroom

The last decade has witnessed a remarkable increase in our ability to measure genetic information. Advancements of sequencing technologies are challenging the existing methods of data storage and analysis. While methods to cope with the data deluge are progressing, many biologists have lagged behind due to the fast pace of computational...

Viral suppressors of RNAI employ a rapid screening mode to discriminate viral RNA from cellular small RNA

RNA interference (RNAi) is an indispensable mechanism for antiviral defense in insects, including mosquitoes that transmit human diseases. To escape this antiviral defense system, viruses encode suppressors of RNAi that prevent elimination of viral RNAs, and thus ensure efficient virus accumulation. Although the first animal Viral Suppressor...

Cas4 Facilitates PAM-Compatible Spacer Selection during CRISPR Adaptation

CRISPR-Cas systems adapt their immunological memory against their invaders by integrating short DNA fragments into clustered regularly interspaced short palindromic repeat (CRISPR) loci. While Cas1 and Cas2 make up the core machinery of the CRISPR integration process, various class I and II CRISPR-Cas systems encode Cas4 proteins for which...

TRBP ensures efficient Dicer processing of precursor microRNA in RNA-crowded environments

The RNA-binding protein TRBP is a central component of the Dicer complex. Despite a decade of biochemical and structural studies, the essential functionality of TRBP in microRNA (miRNA) biogenesis remains unknown. Here we show that TRBP is an integral cofactor for time-efficient Dicer processing in RNA-crowded environments. We competed for...

Single-molecule pull-down for investigating protein–nucleic acid interactions

The genome and transcriptome are constantly modified by proteins in the cell. Recent advances in single-molecule techniques allow for high spatial and temporal observations of these interactions between proteins and nucleic acids. However, due to the difficulty of obtaining functional protein complexes, it remains challenging to study the...