T. Aparicio, V. de Lorenzo, E. Martínez-García, Systems Biology Program, National Center of Biotechnology CSIC, Madrid, 28049 Spain.
Biotechnol J. 2018 May;13(5):e1700161. doi: 10.1002/biot.201700161. Epub 2017 Dec 4.
While adoption of single-stranded DNA recombineering techniques has greatly eased genetic design of the platform strain Pseudomonas putida KT2440, available methods still produce the desired modifications/deletions at low frequencies. This makes isolation of mutants that do not display selectable or conspicuous phenotypes considerably difficult. To overcome this limitation, the authors have merged ssDNA recombineering with CRISPR/Cas9 technology in this bacterium for efficient killing of unmodified cells and thus non-phenotypic selection of bacteria bearing the mutations of interest. After incorporating the system into standardized pSEVA plasmids the authors tested its functional efficiency by targeting different types of changes that ranged from single nucleotide substitutions to one-gene deletions-to even the removal of the large flagellar cluster (≈69 kb). Simultaneous introduction of two independent gene deletions was tested as well. In all cases, directing the crRNA/Cas9 complexes toward non-modified, wild-type genomic sequences boosted dramatically the appearance of the mutants at stake in the absence of any phenotypic selection. The results presented here upgrade the engineering possibilities of the genome of this environmental bacterium (and possibly other Gram-negatives) to obtain modifications that are otherwise cumbersome to generate.
虽然单链 DNA 重组技术的采用极大地简化了平台菌株恶臭假单胞菌 KT2440 的基因设计,但现有的方法仍然以低频率产生所需的修饰/缺失。这使得分离不显示可选择或明显表型的突变体变得相当困难。为了克服这一限制,作者在该细菌中将 ssDNA 重组与 CRISPR/Cas9 技术融合,以有效杀死未修饰的细胞,从而对携带感兴趣突变的细菌进行非表型选择。在将该系统整合到标准化的 pSEVA 质粒中后,作者通过靶向从单个核苷酸取代到一个基因缺失的不同类型的变化,甚至是去除大型鞭毛簇(≈69kb)来测试其功能效率。同时还测试了两个独立基因缺失的同时引入。在所有情况下,将 crRNA/Cas9 复合物引导至未修饰的野生型基因组序列,极大地提高了在没有任何表型选择的情况下处于危险中的突变体的出现率。这里呈现的结果提升了该环境细菌(可能还有其他革兰氏阴性菌)基因组的工程可能性,以获得否则难以生成的修饰。