Efficient Single-Nucleotide Microbial Genome Editing Achieved Using CRISPR/Cpf1 with Maximally 3′-End-Truncated crRNAs
- Authors
- Lee, Ho Joung; Kim, Hyun Ju; Park, Young-Jun; Lee, Sang Jun
- Issue Date
- Jun-2022
- Publisher
- American Chemical Society
- Keywords
- crRNA; FnCpf1; mismatch tolerance; single-base editing
- Citation
- ACS Synthetic Biology, v.11, no.6, pp 2134 - 2143
- Pages
- 10
- Journal Title
- ACS Synthetic Biology
- Volume
- 11
- Number
- 6
- Start Page
- 2134
- End Page
- 2143
- URI
- https://scholarworks.bwise.kr/cau/handle/2019.sw.cau/59649
- DOI
- 10.1021/acssynbio.2c00054
- ISSN
- 2161-5063
- Abstract
- Mismatch tolerance, a cause of the off-Target effect, impedes accurate genome editing with the CRISPR/Cas system. Herein, we observed that oligonucleotide-directed single-base substitutions could be rarely introduced in the microbial genome using CRISPR/Cpf1-mediated negative selection. Because crRNAs have the ability to recognize and discriminate among specific target DNA sequences, we systematically compared the effects of modified crRNAs with 3′-end nucleotide truncations and a single mismatch on the genomic cleavage activity of FnCpf1 inEscherichia coli. Five nucleotides could be maximally truncated at the crRNA 3′-end for the efficient cleavage of the DNA targets of galK and xylB in the cells. However, target cleavage in the genome was inefficient when a single mismatch was simultaneously introduced in the maximally 3′-end-Truncated crRNA. Based on these results, we assumed that the maximally truncated crRNA-Cpf1 complex can distinguish between single-base-edited and unedited targets in vivo. Compared to other crRNAs with shorter truncations, maximally 3′-end-Truncated crRNAs showed highly efficient single-base substitutions (>80%) in the DNA targets of galK and xylB. Furthermore, the editing efficiency for the 24 bases in both galK and xylB showed success rates of 79 and 50%, respectively. We successfully introduced single-nucleotide indels in galK and xylB with editing efficiencies of 79 and 62%, respectively. Collectively, the maximally truncated crRNA-Cpf1 complex could perform efficient base and nucleotide editing regardless of the target base location or mutation type; this system is a simple and efficient tool for microbial genome editing, including indel correction, at the single-nucleotide resolution. ©
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