Molecular Medicine Israel

Type IV-A3 CRISPR-Cas systems drive inter-plasmid conflicts by acquiring spacers in trans


  • •Type IV-A3 CRISPR-Cas acquires spacers using host I-E adaptation machinery
  • •Plasmid-plasmid conflicts involve silencing of plasmid core functions by type IV-A3
  • •DinG helicase is required for repression once transcription has initiated
  • •Type IV-A3 can be programmed as a tool to re-sensitize bacteria to antibiotics


Plasmid-encoded type IV-A CRISPR-Cas systems lack an acquisition module, feature a DinG helicase instead of a nuclease, and form ribonucleoprotein complexes of unknown biological functions. Type IV-A3 systems are carried by conjugative plasmids that often harbor antibiotic-resistance genes and their CRISPR array contents suggest a role in mediating inter-plasmid conflicts, but this function remains unexplored. Here, we demonstrate that a plasmid-encoded type IV-A3 system co-opts the type I-E adaptation machinery from its host, Klebsiella pneumoniae (K. pneumoniae), to update its CRISPR array. Furthermore, we reveal that robust interference of conjugative plasmids and phages is elicited through CRISPR RNA-dependent transcriptional repression. By silencing plasmid core functions, type IV-A3 impacts the horizontal transfer and stability of targeted plasmids, supporting its role in plasmid competition. Our findings shed light on the mechanisms and ecological function of type IV-A3 systems and demonstrate their practical efficacy for countering antibiotic resistance in clinically relevant strains.


CRISPR-Cas systems protect bacteria from invading mobile genetic elements (MGEs) by providing adaptive immunity. Essential to their memory acquisition is the conserved Cas1-Cas2 adaptation module, which excises short sequences (protospacers [PSs]) adjacent to a motif (PAM) in invading MGEs and incorporates them into the CRISPR array as new spacers (Figure 1A). Array transcription is followed by processing into mature CRISPR RNAs (crRNAs), which assemble with Cas proteins into crRNA-guided effector complexes that target and disrupt complementary sequences, typically through nuclease cleavage.1,2

Type IV CRISPR-Cas systems have remained largely understudied, in contrast to most other known CRISPR-Cas types.3 Like other class 1 systems,4 they form multiprotein complexes5,6,7 and are divided into distinct subtypes (IV-A to IV-E) and variants (IV-A1 to IV-A3) based on their molecular architecture.8,9 Although type IV loci contain CRISPR arrays with varying spacer content, they typically lack Cas1-Cas2 adaptation modules, rendering their spacer acquisition mechanism enigmatic (Figure 1A).8,10,11 Type IV CRISPR-Cas systems also stand out for their consistent association with conjugative MGEs, such as plasmids and integrative conjugative elements (ICEs),5,8,10,12 and in case of the type IV-A, for featuring a 5′-3′ DNA helicase called DinG instead of an effector nuclease (Figure 1A).10,13

Recent research has shed light on the potential mechanisms driving RNA-guided type IV CRISPR-Cas targeting. For example, the type IV-A CRISPR-Cas system (variant IV-A1) in Pseudomonas oleovorans has been shown to mediate DinG-dependent transcriptional repression of chromosomal targets.14 Furthermore, type IV-A1 systems can facilitate the loss of a small vector plasmid even when the targeted region is outside an open reading frame,11,14 leaving open questions about the proposed CRISPR interference (CRISPRi) mechanism. Notably, type IV CRISPR arrays are enriched with spacers matching large conjugative plasmids, suggesting a unique role in inter-plasmid conflicts.8,10,12 However, their ecological role and whether and how they can interfere with conjugative plasmids remain unexplored.

Here, through a combination of molecular genetics, bioinformatics, and biochemical analyses, we functionally characterize a type IV-A3 CRISPR-Cas system encoded on a Klebsiella pneumoniae (K. pneumoniae) conjugative plasmid. Our results reveal that type IV-A3 can acquire spacers by co-opting the host-derived type I-E adaptation machinery. Additionally, we show that crRNA-guided targeting can mediate the loss of conjugative plasmids through transcriptional repression of plasmid core functions and demonstrate that this silencing activity can be repurposed to re-sensitize bacteria to antibiotics. Because type IV-A3 systems are widespread among the pervasive and opportunistically pathogenic K. pneumoniae,8,12,15 our findings have important implications for understanding plasmid-driven adaptation, including the prevention and dissemination of antibiotic resistance and virulence factors.

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