Fig. 5: Interfering with Tf1 retrotransposition by CRISPR-Cas13a via targeting its RNA intermediates.

a Schematic representation of three crRNA constructs for Cas13a targeting, for which the crRNA expression cassette was inserted into the Tf1-carrying pHL414 plasmid (as in Fig. 3a) (Supplementary Table S4). Tf1-control contains nontargeting random sequence. b Estimation of Tf1 retrotransposition efficiency by colony-forming assay. Cells were plated in replicates to YES plates with either 5-FOA only or 5-FOA + G418. The values in the upper-left corner indicate the diluted cell concentration (cells/mL), from which 100 μL were plated (Materials and methods). Transposition efficiencies for crRNAs Tf1-835, Tf1-1165 and Tf1-control were estimated by comparing the number of formed colonies between the two plate conditions (Supplementary Data S2). The efficiency values were then normalized with that of Tf1-control as “1%”. Data are means ± SEM (n = 3 independent experiments; *P < 0.05, **P < 0.01 by Student’s t test against Tf1-control). c Growth in liquid culture media (EMM) of LshCas13a strains with either pHL414 plasmid containing Tf1 reporter or pHL414 plasmids containing Tf1 reporter and different crRNA constructs. The strains in the assay were not treated with 5-FOA, for which Cas13a was continually activated in the presence of Tf1 transcripts and targeting crRNAs. The strains containing plasmids Tf1-835, Tf1-1165, crRNA-control or the pHL414 plasmid had an average double time of 5.1, 4.8, 4.8 and 4.7 h, respectively. Data are means ± SEM (n = 4 independent experiments).