Consolidating Ulva functional genomics: Gene editing and new selection systems

The green seaweed Ulva compressa is a promising model for functional biology. In addition to historical research on growth and development, -omics data and molecular tools for stable transformation are available. However, more efficient tools are needed to study gene function. Here, we expand the molecular toolkit for Ulva. We screened the survival of Ulva and its mutualistic bacteria on 14 selective agents and establish that Blasticidin deaminases (BSD or bsr) can be used as selectable markers to generate stable transgenic lines. We show that Cas9 and Cas12a RNPs are suitable for targeted mutagenesis and can generate genomic deletions up to 20 kb using the marker gene ADENINE PHOSPHORIBOSYLTRANSFERASE (APT). We demonstrate that targeted insertion of a selectable marker via homology-directed repair or co-editing with APT is possible for non- marker genes. We evaluated 31 vector configurations and found that bicistronic fusion of Cas9 to a resistance marker or incorporation of introns in Cas9 led to the most mutants. We used this to generate mutants in three non-marker genes using a co-editing strategy. This expanded molecular toolkit now enables us to reliably make gain- and loss-of-function mutants, additional optimizations will be necessary to allow for vector-based multiplex genome editing in Ulva.