Lee Ho Joung, Kim Hyun Ju, Lee Sang Jun
Department of Systems Biotechnology, and Institute of Microbiomics, Chung-Ang University, Anseong 17546, South Korea.
Genome Res. 2020 May;30(5):768-775. doi: 10.1101/gr.257493.119. Epub 2020 Apr 23.
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 in 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 in A single-point nucleotide mutation (T504A or C578A in the gene) was successfully introduced in 36%-95% of negatively selected 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.
CRISPR-Cas9系统彻底改变了基因组编辑。CRISPR-Cas9由单分子引导RNA（sgRNA）和一种蛋白质类Cas9核酸酶组成，该核酸酶识别特定的靶序列和原间隔相邻基序（PAM）序列，随后切割靶向的DNA序列。在位点特异性诱变过程中，这个CRISPR-Cas9系统已被用作一种高效的负选择工具，用于切割未编辑或未改变的靶DNA，从而获得具有所需突变的微生物细胞。本研究旨在调查CRISPR-Cas9系统在细菌体内寡核苷酸定向诱变中的基因组编辑效率。该系统成功地在体内以高编辑效率（81%-86%）引入了两到四个碱基的突变。然而，单点突变（T504A或C578A）很少被引入，编辑效率非常低（<3%），这可能是由于错配耐受性。为了解决这个问题，我们在sgRNA序列中设计了一到两个碱基的错配，以识别体内的靶序列。使用单碱基错配的sgRNA，在36%-95%的负选择细胞中成功引入了单点核苷酸突变（基因中的T504A或C578A）。通过使用错配sgRNA的全基因组单碱基编辑实验随机选择了16个靶点。因此，在48个所需的单碱基突变中，使用错配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。
