CMI：基于 CRISPR/Cas9 的高效多重整合于.

CMI: CRISPR/Cas9 Based Efficient Multiplexed Integration in .

机构信息

Beijing Advanced Innovation Center for Soft Matter Science and Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China.

College of Veterinary Medicine, China Agricultural University, Beijing 100193, China.

出版信息

ACS Synth Biol. 2023 May 19;12(5):1408-1414. doi: 10.1021/acssynbio.2c00591. Epub 2023 Feb 28.

DOI:10.1021/acssynbio.2c00591

PMID:36853024

Abstract

Genomic integration is the preferred method for gene expression in microbial industrial production. However, traditional homologous recombination based multiplexed integration methods often suffer from low integration efficiency and complex experimental procedures. Here, we report a CRISPR/Cas9 based multiplexed integration (CMI) system in , which can achieve quadruple integration at an individual locus without pre-engineering the host. A fused protein, Cas9-Brex27, was used as a bait to attract Rad51 recombinase to the proximity of the double-strand breaks introduced by the CRISPR/Cas9 system. The efficiency of quadruple integration was increased to 53.9% with 40 bp homology arms (HAs) and 78% with 100 bp HAs. CMI was applied to integrate a heterologous mogrol biosynthetic pathway consisting of four genes in a one-step transformation and offered an efficient solution for multiplexed integration. This method expands the synthetic biology toolbox of .

摘要

基因组整合是微生物工业生产中基因表达的首选方法。然而，传统的基于同源重组的多重整合方法往往存在整合效率低和实验步骤复杂的问题。在这里，我们报道了一种基于 CRISPR/Cas9 的多重整合（CMI）系统，可以在不预先对宿主进行工程改造的情况下，在单个基因座上实现四重整合。一种融合蛋白 Cas9-Brex27 被用作诱饵，将 Rad51 重组酶吸引到由 CRISPR/Cas9 系统引入的双链断裂附近。使用 40bp 同源臂（HAs）时，四重整合的效率提高到 53.9%，使用 100bp HAs 时，效率提高到 78%。CMI 被应用于在一步转化中整合由四个基因组成的异源 mogrol 生物合成途径，为多重整合提供了有效的解决方案。该方法扩展了的合成生物学工具包。