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Efficient Single-Nucleotide Microbial Genome Editing Achieved Using CRISPR/Cpf1 with Maximally 3'-End-Truncated
Ho Joung Lee1, Hyun Ju Kim1, Young-Jun Park2
1Department of Systems Biotechnology and Institute of Microbiomics, Chung-Ang University, Anseong 17546, Republic of Korea.
ACS Synthetic Biology
|May 18, 2022
Summary
Maximally truncating CRISPR/Cpf1 crRNAs enhances microbial genome editing accuracy. This modified system efficiently introduces single-base substitutions and indels, improving precision for gene editing applications.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- CRISPR/Cas systems are powerful genome editing tools, but mismatch tolerance causes off-target effects, limiting accuracy.
- CRISPR/Cpf1 systems offer an alternative for genome editing, with crRNA recognition crucial for target specificity.
Purpose of the Study:
- To investigate the impact of crRNA 3'-end truncations and mismatches on CRISPR/Cpf1 cleavage activity.
- To develop a more precise CRISPR/Cpf1-mediated genome editing system for microbial applications.
Main Methods:
- Systematic comparison of modified crRNAs with varying 3'-end nucleotide truncations and single mismatches using FnCpf1 in *Escherichia coli*.
- Evaluation of genomic cleavage activity and editing efficiency at specific target genes (*galK*, *xylB*).
Main Results:
- Maximum cleavage efficiency was achieved with a 5-nucleotide truncation at the crRNA 3'-end.
- Maximally truncated crRNAs enabled highly efficient single-base substitutions (>80%) and single-nucleotide indel introduction (up to 79% in *galK*, 62% in *xylB*).
- The truncated crRNA-Cpf1 complex demonstrated discrimination against mismatched targets, enhancing editing specificity.
Conclusions:
- Maximally 3'-end-truncated crRNAs significantly improve the specificity and efficiency of CRISPR/Cpf1-mediated genome editing.
- This optimized system provides a simple and effective tool for precise microbial genome editing, including base and indel correction at single-nucleotide resolution.
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