Clustered regularly interspaced short palindromic repeats (CRISPR)-associated transposases have the potential to transform the technology landscape for kilobase-scale genome engineering, by virtue of their ability to integrate large genetic payloads with high accuracy, easy programmability and no requirement for homologous recombination machinery. These transposons encode efficient, CRISPR RNA-guided transposases that execute genomic insertions in Escherichia coli at efficiencies approaching ~100%. Moreover, they generate multiplexed edits when programmed with multiple guides, and function robustly in diverse Gram-negative bacterial species. Here we present a detailed protocol for engineering bacterial genomes using CRISPR-associated transposase (CAST) systems, including guidelines on the available vectors, customization of guide RNAs and DNA payloads, selection of common delivery methods, and genotypic analysis of integration events. We further describe a computational CRISPR RNA design algorithm to avoid potential off-targets, and a CRISPR array cloning pipeline for performing multiplexed DNA insertions. The method presented here allows the isolation of clonal strains containing a novel genomic integration event of interest within 1–2 weeks using available plasmid constructs and standard molecular biology techniques.

成簇规则间隔短回文重复序列 (CRISPR) 相关转座酶具有改变千碱基级基因组工程技术格局的潜力，因为它们能够以高精度、易于编程且无需同源重组机制整合大型遗传有效负载。这些转座子编码高效的 CRISPR RNA 引导转座酶，可在大肠杆菌中以接近 100% 的效率执行基因组插入。此外，当使用多个向导进行编程时，它们会生成多重编辑，并在不同的革兰氏阴性细菌物种中发挥强大的功能。在这里，我们提出了使用 CRISPR 相关转座酶 (CAST) 系统工程细菌基因组的详细方案，包括可用载体的指南、指导 RNA 和 DNA 负载的定制、常见传递方法的选择以及整合事件的基因型分析。我们进一步描述了一种计算 CRISPR RNA 设计算法，以避免潜在的脱靶，以及用于执行多重 DNA 插入的 CRISPR 阵列克隆管道。这里介绍的方法允许使用可用的质粒构建体和标准分子生物学技术在 1-2 周内分离包含感兴趣的新型基因组整合事件的克隆菌株。