Endogenous tagging of multiple cellular components in the sea anemone Nematostella vectensis

Abstract

The cnidarian Nematostella vectensis has developed into a powerful model system to study the mechanisms underlying animal development, regeneration, and evolution. However, despite the significant progress in the molecular and genetic approaches in this sea anemone, endogenous protein tagging is still challenging. Here, we report a robust method for knock in for Nematostella using CRISPR/Cas9. As an outcome, we generate endogenously tagged proteins that label core molecular components of several cellular apparatus, including the nuclear envelope, cytoskeleton, cell adhesion, endoplasmic reticulum, cell trafficking, and extracellular matrix. Using live imaging, we monitor the dynamics of vesicular trafficking and endoplasmic reticulum in embryos, as well as cell contractility during the peristaltic wave of a primary polyp. This advancement in gene editing expands the molecular tool kit of Nematostella and enables experimental avenues to interrogate the cell biology of cnidarians.

Optimized husbandry and targeted gene-editing for the cnidarianNematostella vectensis

“…I-SceI protein stimulates homology recombination (HR) by creating a site-specific double strand breaks (DSB) in the genome, in a process called homing (Chames et al, 2005;Silva et al, 2011). Although, CRISPR/Cas9 has also been used to generate genomic insertions in N. vectensis with long homology arms (Ikmi et al, 2014), more recently, a study showed increased efficiency using short homology arms (Paix et al, 2023). These insertions are mainly of two types: large regulatory regions located upstream of coding DNA (i.e.…”

“…These insertions are mainly of two types: large regulatory regions located upstream of coding DNA (i.e. "promoter") followed by a reporter fluorescent protein (He et al, 2018;Ikmi et al, 2014) and gene specific insertion of a reporter fluorescent protein (Gahan et al, 2022;Lebedeva et al, 2022;Lebouvier et al, 2022;Paix et al, 2023). Interestingly, all these CRISPR/Cas9 mediated knock-ins have been generated using sitespecific HR but using two alternative strategies based on long homology arms, about 1Kb long (He et al, 2018;Ikmi et al, 2014;Lebedeva et al, 2022;Lebouvier et al, 2022), or short homology arms, 40-50bp (Gahan et al, 2022;Paix et al, 2023;Seleit et al, 2021).…”

“…"promoter") followed by a reporter fluorescent protein (He et al, 2018;Ikmi et al, 2014) and gene specific insertion of a reporter fluorescent protein (Gahan et al, 2022;Lebedeva et al, 2022;Lebouvier et al, 2022;Paix et al, 2023). Interestingly, all these CRISPR/Cas9 mediated knock-ins have been generated using sitespecific HR but using two alternative strategies based on long homology arms, about 1Kb long (He et al, 2018;Ikmi et al, 2014;Lebedeva et al, 2022;Lebouvier et al, 2022), or short homology arms, 40-50bp (Gahan et al, 2022;Paix et al, 2023;Seleit et al, 2021). Nevertheless, our current study show that similarly to axolotl (Fei et al, 2018), there is also the possibility to generate CRISPR/Cas9 genomic insertions using NHEJ (Figure S3, Figure 5).…”

“…While KOs in N. vectensis are becoming increasingly available, targeted knock-ins remain sparse (reviewed in Röttinger, 2021;Paix et al, 2023). To assay for CRISPR/Cas9-mediated targeted genomic insertion using the newly designed sgRNAs, we assessed if each individual SpCas9/sgRNA complex was able to generate knock-in mutants following a published protocol using HDR (Ikmi et al, 2014).…”

CRISPR/Cas9 knockin methodology for the sea urchin embryo

“…For example, in Medaka, 11% (for mapre1b) to 59% (for g3bp1) of embryos expressed the fluorescent reporter (Seleit et al, 2021). In Nematostella a recent study reported that between 2.2% (for lamin) and 37.7% (for cdh1) of injected embryos were successfully fluorescent (Paix et al, 2023).…”