生物
清脆的
计算生物学
重新调整用途
核糖核酸
整合酶
DNA
遗传学
DNA测序
基因组DNA
基序列
基因
生态学
作者
Zhou‐Hua Cheng,Jie Wu,Jiaqi Liu,Di Min,Dong-Feng Liu,Wen-Wei Li,Han‐Qing Yu
摘要
Abstract Genomic integration techniques offer opportunities for generation of engineered microorganisms with improved or even entirely new functions but are currently limited by inability for efficient insertion of long genetic payloads due to multiplexing. Herein, using Shewanella oneidensis MR-1 as a model, we developed an optimized CRISPR-associated transposase from cyanobacteria Scytonema hofmanni (ShCAST system), which enables programmable, RNA-guided transposition of ultra-long DNA sequences (30 kb) onto bacterial chromosomes at ∼100% efficiency in a single orientation. In this system, a crRNA (CRISPR RNA) was used to target multicopy loci like insertion-sequence elements or combining I-SceI endonuclease, thereby allowing efficient single-step multiplexed or iterative DNA insertions. The engineered strain exhibited drastically improved substrate diversity and extracellular electron transfer ability, verifying the success of this system. Our work greatly expands the application range and flexibility of genetic engineering techniques and may be readily extended to other bacteria for better controlling various microbial processes.
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