Key Laboratory of Molecular Medicine and Biotherapy, Beijing Institute of Technology, School of Life Science, No. 5 South Zhongguancun Street, Beijing 100081, China.
Int J Mol Sci. 2022 Apr 27;23(9):4853. doi: 10.3390/ijms23094853.
is a versatile microbial cell factory that can produce valuable proteins and value-added chemicals. Long fragment editing techniques are of great importance for accelerating bacterial genome engineering to obtain desirable and genetically stable host strains. Herein, we develop an efficient CRISPR-Cas9 method for large-scale and scarless genome engineering in the genome, which can delete up to 134.3 kb DNA fragments, 3.5 times as long as the previous report, with a positivity rate of 100%. The effects of using a heterologous NHEJ system, linear donor DNA, and various donor DNA length on the engineering efficiencies were also investigated. The CRISPR-Cas9 method was then utilized for genome simplification and construction of a series of individual and cumulative deletion mutants, which are further screened for overproducer of isobutanol, a new generation biofuel. These results suggest that the method is a powerful genome engineering tool for constructing and screening engineered host strains with enhanced capabilities, highlighting the potential for synthetic biology and metabolic engineering.
是一种多功能的微生物细胞工厂，可以生产有价值的蛋白质和增值化学品。长片段编辑技术对于加速细菌基因组工程以获得理想的、遗传稳定的宿主菌株非常重要。在此，我们开发了一种高效的 CRISPR-Cas9 方法，用于基因组中的大规模无疤痕基因组工程，该方法可以删除长达 134.3kb 的 DNA 片段，是之前报道的 3.5 倍，阳性率为 100%。还研究了使用异源 NHEJ 系统、线性供体 DNA 和不同供体 DNA 长度对工程效率的影响。然后，将 CRISPR-Cas9 方法用于基因组简化和一系列单个和累积缺失突变体的构建，并进一步筛选新型生物燃料异丁醇的高产菌。这些结果表明，该方法是构建和筛选具有增强能力的工程化宿主菌株的有力基因组工程工具，突出了合成生物学和代谢工程的潜力。
