Genome editing has been revolutionized by the CRISPR-Cas9 system. CRISPR-Cas9 is composed of single-molecular guide RNA (sgRNA) and a proteinaceous Cas9 nuclease, which recognizes a specific target sequence and a protospacer adjacent motif (PAM) sequence and, subsequently, cleaves the targeted DNA sequence. This CRISPR-Cas9 system has been used as an efficient negative-selection tool to cleave unedited or unchanged target DNAs during site-specific mutagenesis and, consequently, obtain microbial cells with desired mutations. This study aimed to investigate the genome editing efficiency of the CRISPR-Cas9 system for in vivo oligonucleotide-directed mutagenesis in bacteria. This system successfully introduced two- to four-base mutations in galK in Escherichia coli with high editing efficiencies (81%-86%). However, single-point mutations (T504A or C578A) were rarely introduced with very low editing efficiencies (<3%), probably owing to mismatch tolerance. To resolve this issue, we designed one- or two-base mismatches in the sgRNA sequence to recognize target sequences in galK in E. coli A single-point nucleotide mutation (T504A or C578A in the galK gene) was successfully introduced in 36%-95% of negatively selected E. coli cells using single-base mismatched sgRNAs. Sixteen targets were randomly selected through genome-wide single-base editing experiments using mismatched sgRNAs. Consequently, out of 48 desired single-base mutations, 25 single bases were successfully edited, using mismatched sgRNAs. Finally, applicable design rules for target-mismatched sgRNAs were provided for single-nucleotide editing in microbial genomes.

中文翻译：

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目标错配的sgRNA辅助CRISPR-Cas9介导的精确微生物基因组编辑。

CRISPR-Cas9系统彻底改变了基因组编辑。CRISPR-Cas9由单分子向导RNA（sgRNA）和蛋白质Cas9核酸酶组成，该酶识别特定的靶序列和原间隔子相邻基序（PAM）序列，然后切割靶DNA序列。该CRISPR-Cas9系统已被用作有效的负选择工具，可在位点特异性诱变过程中裂解未编辑或未修饰的目标DNA，从而获得具有所需突变的微生物细胞。这项研究旨在调查CRISPR-Cas9系统在细菌中体内寡核苷酸定向诱变的基因组编辑效率。该系统成功地在大肠杆菌中以高编辑效率（81％-86％）在galK中引入了2至4个碱基的突变。然而，单点突变（T504A或C578A）很少以极低的编辑效率（<3％）引入，这可能是由于错配耐受性所致。为了解决这个问题，我们在sgRNA序列中设计了一个或两个碱基的错配，以识别大肠杆菌中galK的靶序列。在36％的位点成功地引入了单点核苷酸突变（galK基因中的T504A或C578A） 95％的阴性选择大肠杆菌细胞使用单碱基错配sgRNA。通过使用错配的sgRNA进行全基因组单碱基编辑实验，随机选择了16个靶标。因此，在48个所需的单碱基突变中，使用错配的sgRNA成功编辑了25个单碱基。最后，为微生物基因组中的单核苷酸编辑提供了靶错配sgRNA的适用设计规则。