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 aeruginosa with 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. aeruginosa genome 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, Pseudomonas syringae and Klebsiella pneumoniae, and endogenous CRISPR–Cas use was enhanced with an ‘anti-anti-CRISPR’ strategy. P. aeruginosa Type I-C Cascade–Cas3 (PaeCas3c) facilitates rapid strain manipulation with applications in synthetic biology, genome minimization and the removal of large genomic regions.

CRISPR–Cas 技术已使真核生物和原核生物中的可编程基因编辑成为可能。然而，主要的 Cas9 和 Cas12a 酶在进行大量缺失的能力方面受到限制。在这里，我们使用了进行性核酸酶 Cas3，以及一个基于 IC 型级联的最小系统，用于细菌中的靶向基因组工程。由单个 CRISPR RNA 引导的 DNA 切割在铜绿假单胞菌中产生大的缺失（7-424 kb），效率接近 100%，而 Cas9 产生小缺失和点突变。Cas3 产生源自程序化位点的双向缺失，可用于减少铜绿假单胞菌基因组增加 837 kb (13.5%)。在使用 Cascade–Cas3 而不是 Cas9 进行编辑期间，同源定向修复模板有效地指定了大的删除边界。可转移的“一体式”载体在大肠杆菌、丁香假单胞菌 和肺炎克雷伯菌中发挥作用，并且通过“抗-抗-CRISPR”策略增强了内源性 CRISPR-Cas 的使用。P. aeruginosa Type IC Cascade–Cas3 ( Pae Cas3c) 在合成生物学、基因组最小化和大基因组区域的去除中的应用促进了快速菌株操作。