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

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.

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

Fig. 1. Schematic representation of the CRISPR/Cpf1 system in Corynebacterium glutamicum.

(A) Chromosomal integration of cpf1 in the cg1121 locus via homologous recombination. (B) RecT expression plasmid and crRNA expression plasmid. (C) Scarless genome editing flow. RecT plasmid was electroporated into HK1220 cells. Mutagenic oligonucleotides and crRNA plasmid were electroporated into IPTG-induced HK1220/pHK489 cells. After genome editing, plasmids were cured through culturing at high temperatures and cpf1 was eliminated via the sacB (encoding levansucrase) counter-selection system.

Fig. 2. Carotenoid biosynthesis in C. glutamicum.

(A) Decaprenoxanthin biosynthetic pathway and genes. IPP, isopentenyl pyrophosphate; DMPP, dimethylallyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate. (B) Structure of the crt operon in C. glutamicum ATCC13869.

Fig. 3. Negative selection of oligonucleotide-directed mutations in crtEb by CRISPR/Cpf1.

(A) Negative selection of single-, double-, triple-, and quadruple-base edited targets in crtEb by target-matched CRISPR/Cpf1. Amber stop codon (TAG) generated through oligonucleotide-directed mutagenesis is underlined. (B) The proportion of pink colonies representing the apparent editing efficiency and the number of surviving colonies after electroporation of the target-matched crRNA plasmid (pHK493) and various mutagenic oligonucleotides. Each bar represents the average of three independent experiments. ssODN, single-stranded oligodeoxynucleotide. (C) Schematic representation of the cleavage of single-base-edited targets by CRISPR/Cpf1 owing to mismatch tolerance.

Fig. 4. Single-base genome editing by target-mismatched CRISPR/Cpf1.

(A) Design of target mismatched-crRNAs to prevent the cleavage of single-base-edited DNA targets. The amber stop codon (TAG) generated through oligonucleotide-directed mutagenesis is underlined. (B) The proportion of pink colonies representing the apparent editing efficiency and the number of surviving colonies after electroporation of various target-mismatched crRNA plasmids and single-base-mutagenic oligonucleotides. Each bar represents the average of three independent experiments.

Fig. 5. Sequence alignment of single-base-edited target regions in crtEb.

The PAM sequence of Cpf1 was underlined. Dots and bars indicate perfectly aligned nucleotides and gaps, respectively, in comparison with the target DNA sequence. The gray-shaded nucleotides indicate undesirable mutations. The black-shaded G indicate single-base-edited nucleotides (T150G) after genome editing. E01–E23 show precise single-base changes, and U01–U13 show undesirable substitutions and indels proximal to the edited target region. Parenthesis indicate the proportion of pink colonies among the surviving colonies.