3.1 CRISPR-Cpf1 mediated ldhA knock-out recombination inC. glutamicum.
Cpf1 from Francisella novicida is a putative class 2 CRISPR effector, similar to the role of Cas9 in Streptococcus pyogenes .[34] However, unlike Cas9, Cpf1 cleaves target DNA with a sole single RNA-guided endonuclease and is not coupled to a trans-activating CRISPR RNA (tracrRNA). Additionally, Cpf1 recognizes two or three thymine residues (5’-TTN-3’ or 5’-TTTN-3’) known as the T-rich protospacer-adjacent motif (PAM) and cleaves the phosphodiester bonds between the 23rd and 24th base in the annealing strand and between the 18th and 19th base from the PAM on the non-annealing strand.[15, 34] Although the CRISPR-Cas9 system was confirmed to be a simple and precise tool for genome editing in several microbes, the system does not work well inC . glutamicum . Therefore, the Cpf1 system, adopted in this study, enabled single-strand DNA recombination, endogenous gene deletions, and exogenous gene insertions in C . glutamicum .[15]
To obtain a recombinant C . glutamicum strain with both a gene deletion (lactate dehydrogenase 1, ldhA ) and a gene insertion (kanamycin resistance gene, Knr), the all-in-one, pJYS3_ ΔcrtYF vector was modified to obtain the pJYS3_Amp_MCS vector (Fig. 1). The CRISPR-Cas9 genome editing system has been used previously to modify C. glutamicum . For example, Peng evaluated three factors affecting the recombination efficiency.[13] The authors reported that the recombination efficiency in a gene deletion process was much higher when the length of each homologous arm exceeded 0.1kb, whereas the heterogeneous gene insertion performance improved when the length of a single arm exceeded 0.3 kb. Furthermore, a sgRNA (single guide RNA) including a 20 bp target DNA showed different genome editing efficiencies suggesting that the positions and ratios of the nucleotides in the target DNA sequence must be considered. Therefore, the pJYS3_Amp_DT vector was constructed by incorporating double target DNA sets of the template and non-template strands of the ldhA gene into the pJYS3_Amp_MCS vector. The lengths of the left and right homologous arms were 974 and 942 bp, respectively, and they were subcloned into the pCold vector. A kanamycin resistance gene (1.1 kb) was also sub-cloned between the two homologous arms. The homologous arms and kanamycin resistance gene set (LdhAp-Knr-LdhAt) were introduced into multi-cloning site one of pJYS3_Amp_DT, which resulted in the pJYS3_Amp_DT_(LdhAp-Knr-LdhAt) vector (Fig. 1C). The vector was transformed into C. glutamicumat 46 °C for 6–12 min. The transformants appeared on Knr LB solid media and we confirmed that the first homologous recombination had occurred with a 100% efficiency rate (Fig. 2 and additional file 1: Fig. S1). One colony was picked and suspended in 1 mL BHIS media. After the second heat shock at 46 °C for 6–12 min, appropriately diluted transformant solution was spread on Knr LB media. Twelve colonies out of several transformants were picked and we confirmed that the second homologous recombination occurred with a 50% recombination efficiency (Fig. 2B). Among the transformants, the production of succinic and lactic acids was assessed using line six, a knock-out mutant of the ldhA gene (ΔldhA-6 ), under semi-anaerobic conditions with pure glucose. As expected, the lactic acid production of C. glutamicum was completely blocked (Fig. 2C).