Department of Systems Biotechnology, and Institute of Microbiomics, Chung-Ang University, Anseong, Republic of Korea.
Methods Mol Biol. 2024;2760:147-155. doi: 10.1007/978-1-0716-3658-9_9.
Microbial genome editing can be achieved by donor DNA-directed mutagenesis and CRISPR-Cas12a-mediated negative selection. Single-nucleotide-level genome editing enables the manipulation of microbial cells exactly as designed. Here, we describe single-nucleotide substitutions/indels in the target DNA of E. coli genome using a mutagenic DNA oligonucleotide donor and truncated crRNA/Cas12a system. The maximal truncation of nucleotides at the 3'-end of the crRNA enables Cas12a-mediated single-nucleotide-level precise editing at galK targets in the genome of E. coli.
微生物基因组编辑可以通过供体 DNA 定向诱变和 CRISPR-Cas12a 介导的负选择来实现。单核苷酸水平的基因组编辑可以精确地操纵微生物细胞。在这里，我们使用诱变 DNA 寡核苷酸供体和截断的 crRNA/Cas12a 系统描述了大肠杆菌基因组中靶 DNA 的单核苷酸替换/缺失。crRNA3’-末端核苷酸的最大截断使 Cas12a 能够在大肠杆菌基因组的 galK 靶标上进行单核苷酸水平的精确编辑。
