Fig. 3. Interference of Tf1 retrotransposition by CRISPR-Cas12a.

a Schematic representation of six crRNA constructs for Cas12a targeting, for which the crRNA expression cassette was inserted into the Tf1-carrying pHL414 plasmid (Supplementary Table S4). crNEO-control contains nontargeting random sequence. rrk1 rrk1 promoter, DR direct repeat of crRNA, HDVR hepatitis delta virus ribozyme. b “Transfer-and-patch” assay for detection of Tf1 retrotransposition using plates containing YES media with 5-FOA and G418. The effects of the five different crRNAs and their control crNEO-control were shown side by side. c 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 crRNA 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).