Fig. 1: Sequence-specific bactericidal activity of CRISPR-Cas13a.
a A schematic diagram of transformation of CRISPR-Cas13a and CRISPR-Cas9 with targeting blaIMP-1 into blaIMP-1-expressing E. coli STBL3. b E. coli STBL3 expressing blaIMP-1 from a plasmid (plasmid-borne blaIMP-1) and chromosome (chromosome-borne blaIMP-1) were prepared, and transformed with CRISPR-Cas13a or CRISPR-Cas9, both with spacer targeting blaIMP-1 or no spacer (nontargeting). The resulting transformants were plated on an LB plate containing kanamycin (Km) to test sequence-specific bacterial killing by CRISPR-Cas13a and CRISPR-Cas9. Km was used to maintain the plasmids. c The number of bacteria on the plate obtained in the experiment of b was counted. The statistical significance was determined by two-sided Student’s t-test. Each bar represents the mean with standard deviation (n = 3). d A schematic diagram of the experiment to test CRISPR-Cas13a-dependent cell growth inhibition. Anhydrotetracycline (aTc)-inducible blaIMP-1- or rfp-expression plasmid (pKLC56) was co-transformed with pKLC21 plasmid expressing CRISPR-Cas13a with spacer targeting blaIMP-1 or rfp into E. coli STBL3. The resulting transformants were then cultured and the aTc induction in the presence of the antibiotics Km and Cm (Km and Cm for maintaining plasmids) was carried out at the indicated time points. Thereafter, the OD values were measured every hour. e Growth curves were plotted. Each line of the growth curves represents the mean with standard deviation. f The number of viable cells was counted to calculate the ratio of cell death caused by blaIMP-1-targeting CRISPR-Cas13a. The bacterial culture prepared in d was diluted to 1/100, then aTc was added to induce blaIMP-1. Km and Cm were also added to maintain the CRISPR-Cas13a and the aTc-inducible blaIMP-1 plasmids. Thereafter, the bacterial cultures were sampled at the indicated time points, and the number of surviving bacteria was counted on fresh LB plates. Each bar represents the mean with standard deviation of four biological replicates. p-values were determined by two-sided Student’s t-test. Source data are available in the Source Data file.
