CRISPR-Cas9-mediated pinpoint microbial genome editing aided by target-mismatched sgRNAsopen access
- Authors
- Lee, Ho Joung; Kim, Hyun Ju; Lee, Sang Jun
- Issue Date
- May-2020
- Publisher
- COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
- Citation
- GENOME RESEARCH, v.30, no.5, pp 768 - 775
- Pages
- 8
- Journal Title
- GENOME RESEARCH
- Volume
- 30
- Number
- 5
- Start Page
- 768
- End Page
- 775
- URI
- https://scholarworks.bwise.kr/cau/handle/2019.sw.cau/41782
- DOI
- 10.1101/gr.257493.119
- ISSN
- 1088-9051
1549-5469
- Abstract
- 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 galKgene) 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.
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