Fig. 4. Prolonged crRNA targeting eliminated residual Tf1 retrotransposition by Cas12a.

a Schematic representation of six different crRNA constructs integrated into chromosome II in the six new S. pombe strains. crNEO-control contains nontargeting random sequence. cen2 centromere. 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 the persistent presence of crNEO346 and crNEO-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 crNEO-control as “1%”. Data are means ± SEM (n = 3 independent experiments; **P < 0.01 by Student’s t test against crNEO-control).