Dh5α e coli strain

Each pegRNA was designed to harbor BsaI restriction sites at the 5′ and 3′ end. The single-stranded pegRNAs were amplified to produce double-stranded pegRNAs using Accuprime PFX Supermix, according to the manufacturer’s recommendation (Invitrogen, Waltham, MA, USA). The amplified pegRNAs were digested with BsaI-HF restriction enzyme (New England Biolabs, Ipswich, MA, USA) and ligated into Antarctic phosphatase-treated/BsaI-digested pU6-pegRNA-GG-acceptor plasmid. For constructing the PE3 plasmids, the complementary top and bottom sgRNA strands were purchased separately. Each strand was designed to harbor the BbsI restriction site at the 5′ ends. The 5′ end of each strand was phosphorylated by using the T4 polynucleotide kinase (New England Biolabs) and annealed together. The annealed PE3 was ligated into Antarctic phosphatase-treated/BbsI-digested pKLV-U6-gRNA(BbsI)-PGKHygro2AeGFP. Briefly, the ligation was performed using the T4 ligase (New England Biolabs) at 16 °C overnight, followed by transformation into the chemically competent DH5α E.coli strain (New England Biolabs). The presence of ligated pegRNA and PE3 into their respective expression vectors were verified via Sanger sequencing using U6 promoter Fwd primer.

The complementary sgPDGFB top and bottom strands were purchased separately. These strands were designed to harbor BbsI restriction overhangs at their respective 5′ and 3′ ends for ligation into BbsI-digested pKLV-U6-gRNA(BbsI)-PGKHygro2AeGFP plasmid. The top and bottom strands were annealed and subjected to 5′ end phosphorylation using T4 polynucleotide kinase (New England Biolabs, Ipswich, MA, USA). Ligation was performed using T4 Ligase (New England Biolabs) at 16 °C overnight and the ligated plasmid was transformed into the chemically competent DH5α E. coli strain (New England Biolabs). The presence of sgPDGFB construct within the expression plasmid was verified via Sanger sequencing.

Two gBlock synthetic DNA fragments (IDT) at the lengths of 1.6 and 1.7 kb corresponding to Hyl1La fragments with a 3 × FLAG tag and 20 bp overlaps were ordered and used for generating the expression cassette. These fragments were PCR-amplified by Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs), visualized on 1% agarose gel and purified by NucleoSpin Gel and PCR Clean-up (Macherey-Nagel). Gibson assembly was performed with the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) following the manufacturer’s protocol. The resulting product was further subcloned by restriction digestion with AscI and SalI into a pER242 vector having a TBP promoter previously proved to drive ubiquitous expression in Nematostella (Admoni et al., 2020 (link)), memOrange2, and SV40 polyadenylation signal (Figure 5A). The transformation was performed in E. coli DH5α strain (New England Biolabs). The plasmid was purified by HiSpeed Plasmid Midi Kit (Qiagen, Hilden, Germany) and sequenced by the Sanger method (HyLabs, Israel); 100 ng/µl of purified plasmid was injected into the fertilized Nematostella embryo and visualized after 2 days under an SMZ18 stereomicroscope equipped with a DS-Qi2 camera (Nikon, Tokyo, Japan).