Directed evolution and targeted genome editing have been deployed to create genetic variants with usefully altered phenotypes. However, these methods are limited to high-throughput screening methods or serial manipulation of single genes. In this study, we implemented multicopy chromosomal integration using CRISPR-associated transposases (MUCICAT) to simultaneously target up to 11 sites on the Escherichia coli chromosome for multiplex gene interruption and/or insertion, generating combinatorial genomic diversity. The MUCICAT system was improved by replacing the isopropyl-beta-D-thiogalactoside (IPTG)-dependent promoter to decouple gene editing and product synthesis and truncating the right end to reduce the leakage expression of cargo. We applied MUCICAT to engineer and optimize the N-acetylglucosamine (GlcNAc) biosynthesis pathway in E. coli to overproduce the industrially important GlcNAc in only 8 days. Two rounds of transformation, the first round for disruption of two degradation pathways related gene clusters and the second round for multiplex integration of the GlcNAc gene cassette, would generate a library with 1–11 copies of the GlcNAc cassette. We isolated a best variant with five copies of GlcNAc cassettes, producing 11.59 g/L GlcNAc, which was more than sixfold than that of the strain containing the pET-GNAc plasmid. Our multiplex approach MUCICAT has potential to become a powerful tool of cell programing and can be widely applied in many fields such as synthetic biology.

中文翻译：

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使用 CRISPR 相关转座酶通过多拷贝染色体整合对细胞进行编程

定向进化和靶向基因组编辑已被用于创建具有有用改变表型的遗传变异。然而，这些方法仅限于高通量筛选方法或单基因的串行操作。在这项研究中，我们使用 CRISPR 相关转座酶 (MUCICAT) 实现了多拷贝染色体整合，以同时靶向大肠杆菌上的多达 11 个位点染色体用于多重基因中断和/或插入，产生组合基因组多样性。MUCICAT 系统通过替换异丙基-β-D-硫代半乳糖苷 (IPTG) 依赖性启动子来改进基因编辑和产品合成，并截断右端以减少货物的泄漏表达。我们应用 MUCICAT 来设计和优化大肠杆菌中的 N-乙酰氨基葡萄糖 (GlcNAc) 生物合成途径仅在 8 天内过量生产具有工业重要性的 GlcNAc。两轮转化，第一轮用于破坏与两个降解途径相关的基因簇，第二轮用于 GlcNAc 基因盒的多重整合，将生成具有 1-11 个 GlcNAc 盒拷贝的文库。我们分离了一个具有 5 个 GlcNAc 盒拷贝的最佳变体，产生 11.59 g/L GlcNAc，是含有 pET-GNAc 质粒的菌株的六倍多。我们的多重方法 MUCICAT 有潜力成为细胞编程的强大工具，可以广泛应用于合成生物学等许多领域。