Design of a versatile pH-regulated CrisPR-Cas9 plasmid for plasmid curing and chromosome targeting in Lactococcus lactis

GARAY NOVILLO, J. N.; RUIZ MASÓ, J. A.; DEL SOLAR, G.; BARRA, J. L.

Congreso; SAIB - SAMIGE Joint meeting 2021 on line; 2021

To date CRISPR-Cas9 technology for the editing or removal of chromosomal or extra-chromosomal genetic elements in somespecies of lactic acid bacteria (LAB) have yet to improve. In this work, we propose a single plasmid strategy to modify a LABstrain and lastly obtain a plasmid free strain with the desired modification. We have constructed the versatile pILCC9 vectorexpressing the Cas9 nuclease gene under two different pH-regulated promoters, and the DNA encoding the scaffold domainof a single guide RNA (sgRNA) under the control of a strong constitutive promoter. pILCC9 is a shuttle vector containing alow copy number replicon functional in Escherichia coli, which permits the construction of the different plasmid derivatives,and a theta-type promiscuous replicon, which is able to replicate in a wide range of LAB. Vector pILCC9 can be used to clonea DNA fragment encoding a specific targeting domain (spacer) of the sgRNA, which enables the Cas9-mediated generation ofdouble-strand DNA breaks (DSB) within chromosomal or mobile genetic element targets. Transformation of the model LABLactococcus lactis MG1363 with any of these plasmid derivatives yielded transformation efficiencies of ~104cfu/μg.Incubation of transformants containing the chromosome-targeting sgRNA in GM17 adjusted to acidic pH or buffered GM17led to strong cell lethality. When the sgRNA was directed to a plasmid target, growing of transformants, previously carryingdifferent plasmids, in acidic GM17 resulted in a complete elimination of the targeted vector in almost all cells. This assay wastested for low and high copy number plasmids resulting both in successful elimination. Our results showed that the CRISPRCas9system can be pH-controlled and considering the lactic acid production of L. lactis, it could be either autoinduced oncethe culture gained biomass, or externally induced using acid media, to efficiently direct a chromosomic or plasmidic DSB.Last of all, our results showed that the CRISPR-Cas9 plasmid, pILCC9, replicating in L. lactis exhibits a relatively highplasmid loss rate after growing in the absence of selective pressure, which guarantees a plasmid free strain once modified.