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CRISPR–Cas technologies have enabled programmable gene editing in eukaryotes and prokaryotes. However, the leading Cas9 and Cas12a enzymes are limited in their ability to make large deletions. Here, we used the processive nuclease Cas3, together with a minimal Type I-C Cascade-based system for targeted genome engineering in bacteria. DNA cleavage guided by a single CRISPR RNA generated large deletions (7–424?kilobases) in Pseudomonas aeruginosawith near-100% efficiency, while Cas9 yielded small deletions and point mutations. Cas3 generated bidirectional deletions originating from the programmed site, which was exploited to reduce the P. aeruginosagenome by 837?kb (13.5%). Large deletion boundaries were efficiently specified by a homology-directed repair template during editing with Cascade–Cas3, but not Cas9. A transferable ‘all-in-one’ vector was functional in Escherichia coli, Pseudomonassyringaeand Klebsiella pneumoniae, and endogenous CRISPR–Cas use was enhanced with an ‘anti-anti-CRISPR’ strategy. P. aeruginosaType I-C Cascade–Cas3 (PaeCas3c) facilitates rapid strain manipulation with applications in synthetic biology, genome minimization and the removal of large genomic regions.

Pathogens use effector proteins to suppress host immunity and promote infection. However, plants can recognize specific effectors and mount an effector-triggered immune response that suppresses pathogen growth. The YopJ/HopZ family of type III secreted effector proteins is broadly distributed in bacterial pathogens of both animals and plants. These effectors can either suppress host immunity or elicit defense responses depending on the host genotype. In a recent report, we identified an Arabidopsis thalianapseudokinase ZED1 that is required for the recognition of the Pseudomonas syringaeHopZ1a effector. Here we discuss the role of ZED1 in HopZ1a recognition, and present models of effector recognition in plants. We draw parallels between HopZ1a and YopJ effector proteins, and between ZED1 and other immunity-related kinases that can be targeted by pathogen effectors.