U6 promoters in Ulva prolifera enable dual small-RNA/mRNA transcription and vector-based CRISPR-Cas9 genome editing

For establishing vector-based gene editing in the green-tide-forming alga Ulva prolifera, development of endogenous RNA polymerase III (Pol III) promoters is a prerequisite to drive single-guide RNA (sgRNA) transcription. To meet the need, the promoters of U6 snRNA genes in U. prolifera (UpU6) were identified, cloned, and functionally characterized. Results showed that three UpU6 copies shared identical sequences, with divergent 5' upstream sequences containing a positionally conserved TATA box but lacking the USE element typical of seed plants. All three UpU6 promoters were shown to be capable of driving not only small RNA transcription but also protein-coding gene expression in U. prolifera, exhibiting the dual transcriptional activity. Unexpectedly, all three UpU6 promoters also supported GUS reporter gene expression in Agrobacterium, representing the first demonstration of U6 promoter activity in a prokaryotic system. The results of the gene-editing experiment showed that, all three UpU6 promoters supported vector-based CRISPR-Cas9 targeting of the APT gene, yielding mutant strains. The mutation-positive fraction among resistant thalli was 92.3% (48/52), and the editing frequency calculated from the estimated starting cell number averaged 3.26 × 10⁻⁶ across the three UpU6 promoters and did not differ significantly among them. In the mutants, high-frequency occurrences of base substitutions (45.5%) and insertions of long Mutator-like element (MULE) (18.2%) were detected, highlighting a potential association between endogenous DNA transposon insertion and CRISPR-Cas9-mediated gene editing. These findings expanded understanding of eukaryotic U6 promoter functionality and enrich the toolkit for vector-based CRISPR-Cas9 genome editing in U. prolifera.