Miniature CRISPR-Cas12f1-Mediated Single-Nucleotide Microbial Genome Editing Using 3 '-Truncated sgRNAopen access
- Authors
- Lee, Ho Joung; Kim, Hyun Ju; Lee, Sang Jun
- Issue Date
- Feb-2023
- Publisher
- MARY ANN LIEBERT, INC
- Citation
- CRISPR JOURNAL, v.6, no.1, pp 52 - 61
- Pages
- 10
- Journal Title
- CRISPR JOURNAL
- Volume
- 6
- Number
- 1
- Start Page
- 52
- End Page
- 61
- URI
- https://scholarworks.bwise.kr/cau/handle/2019.sw.cau/60596
- DOI
- 10.1089/crispr.2022.0071
- ISSN
- 2573-1599
2573-1602
- Abstract
- The CRISPR-Cas system has been used as a convenient tool for genome editing because the nuclease that cuts the target DNA and the guide RNA that recognizes the target are separated into modules. Cas12f1, which has a smaller size than that of other Cas nucleases, is easily loaded into vectors and is emerging as a new genome editing tool. In this study, AsCas12f1 was used to negatively select only Escherichia coli cells obtained by oligonucleotide-directed genome editing. Although double-, triple-, and quadruple-base substitutions were accurately and efficiently performed in the genome, the performance of single-base editing was poor. To resolve this limitation, we serially truncated the 3 '-end of sgRNAs and determined the maximal truncation required to maintain the target DNA cleavage activity of Cas12f1. Negative selection of single-nucleotide-edited cells was efficiently performed with the maximally 3 '-truncated sgRNA-Cas12f1 complex in vivo. Moreover, Sanger sequencing showed that the accuracy of single-nucleotide substitution, insertion, and deletion in the microbial genome was improved. These results demonstrated that a truncated sgRNA approach could be widely used for accurate CRISPR-mediated genome editing.
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Collections - College of Biotechnology & Natural Resource > Department of Systems Biotechnology > 1. Journal Articles
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