Fig. 3.

Application and improvement of CRISPR/Cas9 system in L. lactis NZ9000. a The nucleotide sequences of protospacers and PAM sites in the four genomic locations to be targeted. b The targeting efficiency by the low copy number vector pTRKL₂ based CRISPR/Cas9 system. When transforming NZ9000 with pTLCas9galk1 (or pTLCas9hemN1, pTLCas9recA1, pTLCas9noxD1), there was a 10⁵-fold reduction in viable colonies compared to transformation with pTLCas9 (control). c Reduction of the off-target effects in L. lactis. The Cas9, tracrRNA and crRNA were carried by the high copy number plasmid pTRKH₂. The spacer sequence was set as 20 bp, 25 bp and 30 bp for each of the four genomic locations. Statistically significant differences are indicated by asterisks (*P < 0.05; **P < 0.01). The values are means ± standard deviations of three independent experiments