# Interdependent Transcription of a Natural Sense/Antisense Transcripts Pair (_SLC34A1_/Pfn3)
## 2 Results
### Genetic Interference with SLC34A1/PFN3 Sense-Antisense Expression  
To scrutinize this hypothesis, we aimed to modulate _SLC34A1/PFN3_ sense and antisense transcription individually by means of CRISPR-Cas9-mediated insertion of transcriptional termination cassettes. HEK293 cells were used for the knock-in of a transcription termination cassette. Guide RNAs were designed to direct Cas9 to the second (sense) and the first intron (antisense), respectively (Figure 5A). Cassettes were prepared containing the CMV promoter driving a selection marker that confers resistance to the antibiotic puromycin in addition to a strong polyadenylation signal (BGH) and specific sequences flanking the double strand breaks to direct homologous DNA repair (Figure 5B) [31]. Biallelic insertions were achieved with a single homology template, thanks to highly efficient Cas9. The presence of both mono- and bi-allelic insertions in individual clones was tested by PCR (Figure S3), and clones with bi-allelic insertion were used for further studies. First, the consequences of knock-ins on _SLC34A1/PFN3_ expression were tested using various primer pairs downstream of the insertion sites. Unexpectedly, significant transcript levels could be detected for sense (S2 and S3 in the middle panel of Figure 5C) and antisense (AS2 in the right panel of Figure 5D), indicating that the promoter driving the antibiotic resistance gene produced read-through transcripts into the opposite gene. Intriguingly, the read-through transcripts provided a tool to differentiate between transcriptional activation by dexamethasone and transcriptional interference, where transcription in one direction alters the RNA levels of the complementary transcript. The level of _SLC34A1_ read-through RNA was 7-10 times (Figure 5C middle, S2 and S3), the _PFN3_ truncated transcript 3-5 times (Figure 5D, right panel) over the background of non-edited, non-stimulated HEK293 cells. The small difference in sense and antisense transcripts may reflect different inherent promoter activity of sense/antisense or the stability of the two RNAs. Importantly, the level of read-through transcripts mirrors the increased expression of sense and antisense RNAs in response to dexamethasone. The consequences of the read-though transcripts on RNA levels from the opposite gene were tested by RT-qPCR: Transcription from either direction leads to a significant threefold increase in the respective complementary mRNAs (antisense-induced sense expression, Figure 5C right panel; sense-induced antisense expression, Figure 5D, middle panel). Comparable experiments were performed with knock-in cells after exposure to dexamethasone. However, the effects of the drug were almost completely blunted by the presence of the cassette, regardless of the direction of its transcription. To test whether the positive interference between sense and antisense transcript was acting in cis, we transiently transfected HEK293 cells with PCR fragments encoding each of the two transcripts (50 and 250 ng/mL). At 50 ng/mL, neither sense nor antisense transgene stimulated the expression of the opposite transcript (Figure 6). The higher amount of DNA appeared to over-saturate the cells since the expression of transgene was not further increased compared to the lower dose. The complementary transcript appeared to become stabilized, though this effect may be unspecific. Figure 5: Constitutive low-level activation of sense and antisense expression in HEK293 cells. (**A**) Snapshot of the _SLC3 AA1/PFN3_ locus with the insertion sites for the HDR (homology-directed repair) cassettes and primer sites S1–S3 as well as AS1 and AS2. (**B**) Schematic representation of the HDR cassette containing a CMV promoter, the puromycin resistance, the BGH polyadenylation signal and the two gene-specific flanking regions (homologous arm). The cassette was meant to shut down transcription, but insertion produced low levels of read-through transcripts driven by the CMV promoter.