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Single-Base Genome Editing in Corynebacterium glutamicum with the Help of Negative Selection by Target-Mismatched CRISPR/Cpf1

Journal of Microbiology and Biotechnology 2020년 30권 10호 p.1583 ~ 1591
김현주, 오세영, 이상준,
소속 상세정보
김현주 ( Kim Hyun-Ju ) - Chung-Ang University Department of Systems Biotechnology
오세영 ( Oh Se-Young ) - Chung-Ang University Department of Systems Biotechnology
이상준 ( Lee Sang-Jun ) - Chung-Ang University Department of Systems Biotechnology

Abstract


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 Corynebacterium glutamicum. This study aimed to introduce an amber stop codon in crtEb encoding lycopene hydratase, through ODM and Cpf1-mediated negative selection; deficiency of this enzyme causes pink coloration due to lycopene accumulation in C. glutamicum. Consequently, on using double-, triple-, and quadruple-basemutagenic oligonucleotides, 91.5?95.3% pink cells were obtained among the total live C. glutamicum 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 C. glutamicum.

키워드

CRISPR/Cpf1; Corynebacterium glutamicum; single-base genome editing; target-mismatched crRNA; mismatch tolerance

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