Fig. 2

CRISPR/Cas9 as counterselection marker after ssDNA recombineering. a Sequence of the targeted upp region of L. lactis NZ9000 is shown aligned with amino acids specified by each codon. If the desired mutation (red bases) is introduced, two successive stop codons will be produced. b Overview of ssDNA recombineering coupled CRISPR–Cas9 counterselection. The bases of PAM are underlined. In the first 36 h, competent L. lactis cells expressing recombinase RecT was prepared, and transformed with ssDNA uppo and pTHCas9upp simultaneously by electroporation. After transformation, the cells were recovered in GM17 agar containing erythromycin for 24 h. In the second 36 h, the colonies were picked up, and the expected mutation was PCR amplification and subjected to sequencing analysis. c Re-screening of the recombineered colonies using 5-fluorouracil. The Δupp mutants containing desired mutations were capable of growing on GM9 agar containing 10 μg/mL 5-fluorouracil, but the WT was unable to grow. GM9 agar was used as a control. d Sequencing of the Δupp mutant. The bases in the red box are mutations