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. 2024 Jul 31;16(8):evae166. doi: 10.1093/gbe/evae166

Fig. 2.

Fig. 2.

Deletion of tRNA-Leu(CAG) in S. capsularis is viable. a) Scheme for deletion of the tRNA-Leu(CAG) gene of S. capsularis NRRL Y-17639 by CRISPR-Cas9 RNPs. The repair template containing a kanamycin resistance cassette (KanMX, modified to lack CUG codons) was amplified from a plasmid using primers with 50-nucleotide tails to generate homology arms matching the regions upstream and downstream of the tRNA-Leu(CAG) gene. CRISPR RNPs containing a guide RNA targeting exon 2 of the tRNA-Leu(CAG) gene were assembled in vitro and electroporated into competent cells. Transformants were screened by junction PCR using primer pairs 3/4 and 5/6 (EOC29/EOC1 and EOC3/EOC30). b) PCR confirmation of three tRNA-Leu(CAG) deletion strains, by amplification of the entire recombinant locus with primer pair 3/6 (EOC29/EOC30). The three lanes marked PCR are transformant colonies with the deletion; WT is a wild-type NRRL Y-17639 control; N is a negative control; and M is a size marker ladder. c) Growth curves comparing the nine independent tRNA-Leu(CAG) deletion strains (red), to three biological replicates of the wild-type strain (blue), at 25 °C and 37 °C. Median values at each time point are shown in the darker shade.