Validation of CRISPR-based MS2 knockin in the clonal cells. (A, C, and E) Schematic diagram of primer designs to validate the insertion of MS2V5_17× into rDNA at the regions of 3′-ETS (A, three independent clones), ITS1 (C, one clone), and 5.8S (E, one clone) in clonal cells, respectively. sgRNA design for CRISPR knockin is also highlighted to illustrate the insertion site for each region. Representative gels of PCR products are shown to indicate the correct insertions. An unspecific band amplified from 3′-ETS clone is pointed out by asterisk (A, right). The size of all PCR products is correct except the fragment amplified from 3′-ETS clone 2 or ITS1 clone (using F3 and R2 primers), which is shorter than expected due to a deletion in this region revealed by DNA sequencing. Notably, MS2V5-3′HA junction could not be successfully amplified, which might be due to the high GC content (∼80%) in 3′-ETS. (B, D, and F) Example chromatogram showing successful recombination for each insertion site at 3′-ETS (B), ITS1 (D), and 5.8S (F). The junction between homology arm and MS2 sequence was shown to indicate the correct insertion. Source data are available for this figure: SourceData FS2.