biostudies-literatureKim HJCJ CheilJedang Institute of BiotechnologyChung-Ang University1583-1591https://www.ebi.ac.uk/biostudies/studies/S-EPMC9728170biostudies-literatureUnknown30(10)CRISPR/Cpf1 has emerged as a new CRISPR-based genome editing tool because, in comparison with CRIPSR/Cas9, it has a different T-rich PAM sequence to expand the target DNA sequence. Single-base editing in the microbial genome can be facilitated by oligonucleotide-directed mutagenesis (ODM) followed by negative selection with the CRISPR/Cpf1 system. However, single point mutations aided by Cpf1 negative selection have been rarely reported in <i>Corynebacterium glutamicum</i>. This study aimed to introduce an amber stop codon in <i>crtEb</i> encoding lycopene hydratase, through ODM and Cpf1-mediated negative selection; deficiency of this enzyme causes pink coloration due to lycopene accumulation in <i>C. glutamicum</i>. Consequently, on using double-, triple-, and quadruple-basemutagenic oligonucleotides, 91.5-95.3% pink cells were obtained among the total live <i>C. glutamicum</i> cells. However, among the negatively selected live cells, 0.6% pink cells were obtained using single-base-mutagenic oligonucleotides, indicating that very few single-base mutations were introduced, possibly owing to mismatch tolerance. This led to the consideration of various targetmismatched crRNAs to prevent the death of single-base-edited cells. Consequently, we obtained 99.7% pink colonies after CRISPR/Cpf1-mediated negative selection using an appropriate singlemismatched crRNA. Furthermore, Sanger sequencing revealed that single-base mutations were successfully edited in the 99.7% of pink cells, while only two of nine among 0.6% of pink cells were correctly edited. The results indicate that the target-mismatched Cpf1 negative selection can assist in efficient and accurate single-base genome editing methods in <i>C. glutamicum</i>.Journal of microbiology and biotechnologySingle-Base Genome Editing in <i>Corynebacterium glutamicum</i> with the Help of Negative Selection by Target-Mismatched CRISPR/Cpf1.PMC9728170CG-20-17-01-0002Oh SYLee SJKim HJfalseSingle-Base Genome Editing in <i>Corynebacterium glutamicum</i> with the Help of Negative Selection by Target-Mismatched CRISPR/Cpf1.CRISPR/Cpf1 has emerged as a new CRISPR-based genome editing tool because, in comparison with CRIPSR/Cas9, it has a different T-rich PAM sequence to expand the target DNA sequence. Single-base editing in the microbial genome can be facilitated by oligonucleotide-directed mutagenesis (ODM) followed by negative selection with the CRISPR/Cpf1 system. However, single point mutations aided by Cpf1 negative selection have been rarely reported in <i>Corynebacterium glutamicum</i>. This study aimed to introduce an amber stop codon in <i>crtEb</i> encoding lycopene hydratase, through ODM and Cpf1-mediated negative selection; deficiency of this enzyme causes pink coloration due to lycopene accumulation in <i>C. glutamicum</i>. Consequently, on using double-, triple-, and quadruple-basemutagenic oligonucleotides, 91.5-95.3% pink cells were obtained among the total live <i>C. glutamicum</i> cells. However, among the negatively selected live cells, 0.6% pink cells were obtained using single-base-mutagenic oligonucleotides, indicating that very few single-base mutations were introduced, possibly owing to mismatch tolerance. This led to the consideration of various targetmismatched crRNAs to prevent the death of single-base-edited cells. Consequently, we obtained 99.7% pink colonies after CRISPR/Cpf1-mediated negative selection using an appropriate singlemismatched crRNA. Furthermore, Sanger sequencing revealed that single-base mutations were successfully edited in the 99.7% of pink cells, while only two of nine among 0.6% of pink cells were correctly edited. The results indicate that the target-mismatched Cpf1 negative selection can assist in efficient and accurate single-base genome editing methods in <i>C. glutamicum</i>.2020-01-01T00:00:00Z2020 Oct2025-04-04T22:15:05.129Z2025-04-04T22:15:05.129ZS-EPMC97281703280775610.4014/jmb.2006.06036