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

JAVIER NICOLÁS GARAY NOVILLO; RUIZ-MASÓ, JOSÉ ÁNGEL; DEL SOLAR, GLORIA; BARRA, JOSÉ LUIS

Online

Congreso; LVII SAIB - XVI SAMIGE Annual Joint meeting 2021 on line; 2021

SAIB-SAMIGE

To date CRISPR-Cas9 technology for the editing or removal of chromosomal or extra-chromosomal genetic elements in some species of lactic acid bacteria (LAB) have yet to improve. In this work, we propose a single plasmid strategy to modify a LAB strain and lastly obtain a plasmid free strain with the desired modification. We have constructed the versatile pILCC9 vector expressing the Cas9 nuclease gene under two different pH-regulated promoters, and the DNA encoding the scaffold domain of a single guide RNA (sgRNA) under the control of a strong constitutive promoter. pILCC9 is a shuttle vector containing a low 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 clone a DNA fragment encoding a specific targeting domain (spacer) of the sgRNA, which enables the Cas9-mediated generation of double-strand DNA breaks (DSB) within chromosomal or mobile genetic element targets. Transformation of the model LAB Lactococcus 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 GM17 led to strong cell lethality. When the sgRNA was directed to a plasmid target, growing of transformants, previously carrying different plasmids, in acidic GM17 resulted in a complete elimination of the targeted vector in almost all cells. This assay was tested for low and high copy number plasmids resulting both in successful elimination. Our results showed that the CRISPR-Cas9 system can be pH-controlled and considering the lactic acid production of L. lactis, it could be either autoinduced once the 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 high plasmid loss rate after growing in the absence of selective pressure, which guarantees a plasmid free strain once modified.