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

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.

Repair donors were synthesized by PCR using primers (Sigma, salt-free, 100μM in H2O) annealing with the extremities of the inserts and contain sequence homologous to each side of the Cas9 cut (homology arms) and if applicable, sequence between Cas9 cut and insertion site (2). If necessary, silent mutations were introduced to reduce the Cas9 cut of the donor / edited locus, and to avoid homology between the sequence from the insert and the Cas9 cut with the genomic sequence. Guide RNAs were selected to cut as close as possible to the site of insertion and to be predicted as efficient sgRNA (5). PCR mix was: 0.8µl of plasmid template at 50-100ng/µl (standard plasmid miniprep), 2µl of each PCR primers at 100µM, 200µl of 2X Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB, #M0531L), 195.2µl of H2O, and split in 8*50µl in PCR tubes. PCR condition were 98°C / 30s at 98°C, 30s at annealing temperature, 45s at 72°C, for 30 cycles / 10 minutes at 72°C / Hold at 10°C. Annealing temperature was 61.5°C for most PCR repair donors, but sometimes a gradient from 60 °C to 72 °C was performed to obtain single bands on agarose gel. PCR amplicons were pooled and purified together using MinElute PCR Purification Kit (Qiagen, #28004) and eluted in 10µl of H2O. Concentration was measured using a NanoDrop.
After injection, embryos were kept at 17 °C overnight. The day after, they were gently washed twice with 1/3-SW and kept at 17 °C for another day. The following day, embryos were transferred to a new petri dish with 1/3-SW and kept at 27 °C. At day three following injection, injected embryos were selected for positive dextran fluorescence and transferred to a new dish containing 1/3-SW. Positive KI were identified by screening for the corresponding fluorescence in the following days, either live using a Nikon SMZ18 stereomicroscope with appropriate filters; or fixed, mounted, and screened with a confocal microscope. Numbers are (positive F0s / total injected): lamin KI ( For double fluorescent color KI of mhc, the injection mix contained each repair donor at half the concentration of what was used for single color KI. Injection dye was 1μl FITC (Invitrogen, #F1300, 0.1mg/ml in Tris 5mM pH7.5) and 1μl dextran A680 (Invitrogen, #D34680, 50ug/µl in H2O). Due to the transient labeling of FITC and the difficulty of identifying dextran A680 embryos, injected embryos were not sorted. Numbers are (among KI positive F0s): 11/33 mNG+/mScarlet+, 11/33 mNG+/mScarlet-, 11/33 mNG-/mScarlet+ (first experiment) and 5/18 mNG+/mScarlet+, 8/18 mNG+/mScarlet-, 5/18 mNG-/mScarlet+ (second experiment).
KI positive F0s were grown to adulthood and crossed with wild-type animals to determine germline transmission.

Imaging
Embryos were collected and transferred to a well of a 96 well plate containing 1/3-SW. When necessary, primary polyps were anesthetized for 30m-1h by gently adding MgCl2 solution to the 1/3-SW (from a stock solution at 7% w/v of MgCl2 in 1/3-SW, 0.35% final). Next, 1/3-SW (with or without MgCl2) was replaced by a solution of 3 parts of 1X PBS and 1 part of PFA 16% (Pierce, #28908). Fixation was performed for 1-2h at RT with occasional mixing by pipetting. Next, fixed embryos were washed once with 1X PBS, followed by a wash with 1X PBS containing 0.25% Triton X-100, and finally 3 times with 1X PBS. Fixed embryos were transferred to a microscope slide coated with Poly-L-lysine solution (Sigma-Aldrich, #P8920) inside a square of double side tape. Excess 1X PBS was removed, and a drop of VectaShield Antifade Mounting Medium with DAPI (Biozol, #VEC-H-1200-10) was added. Next, a glass coverslip was added, and the slide was sealed with CoverGrip (Biozol, #BOT-23005).
Imaging was performed with a Leica SP8 confocal or a Zeiss LSM880 with appropriate settings for eGFP/mNG and mScarlet imaging. For whole embryos imaging at low resolution, a Biorad Zoe fluorescent microscope was also used. For live imaging of the mhc::mNG line, a mesoscopic oblique plane microscope was used to generate maximum intensity projection (600 z slices, 1.3µm apart with 1ms exposure time and 2mW excitation power) (7). For live imaging of mNG::sec61b and eGFP::rab11a lines, a Zeiss LSM980 Airy Fast microscope was used. Images were processed using ImageJ Fiji and eGFP::Rab11a particles were tracked using Manual Tracking plug-in.

Genotyping and Sequencing
Individual F1s primary polyps were transferred first in a dish containing H2O, and next in PCR tubes. Next, they were lysed in 30μl of QuickExtract DNA Extraction Solution (VWR, #BZYM101098) for 3h at 65 °C (with time-to-time vortexing and spinning), followed by 30min at 100 °C. Finally, 30μl of H2O was added to the lysis, and samples were vortex and stored at -20 °C. Alternatively, adult animals were anesthetized with 0.35% MgCl2, the tentacles were cut and transferred in a dish containing H2O, and next in Eppendorf tubes, spun for 2m at 13000rpm in a microcentrifuge and processed as for individual primary polyps.
For genotyping PCR (with primers to amplify the targeted locus, the 5'/3' insertion junctions, or the fluorescent reporter sequence), the reaction was set up as it was for PCR repair donor, but with 4μl of lysis (2μl for tentacle samples) and a final volume of 20μl. Detection of possible imprecise insertion at the targeted locus, or random insertion of the fluorescent reporter, or formation of long lasting episome, was performed by PCR using fluorescent reporter specific primers on fluorescent-negative F1s (from broods containing also fluorescent-positive F1s). Fluorescent-negative F1s were also sequenced at the targeted locus and NHEJ alleles were identified by the presence of a mix of two sequences in Sanger sequencing (wildtype + NHEJ).
For each primer pair, the best annealing temperature was determined using a temperature gradient from 60 °C to 72 °C, using similar PCR conditions as for synthesis of PCR donors. PCR amplicons were analyzed on agarose gels and imaged using standard UV transilluminator or Typhoon imager (GE Healthcare). PCR amplicons were column purified using QIAquick PCR Purification Kit (Qiagen, #28104), eluted in 30µl H2O, and sent out for Sanger sequencing (Eurofins Genomics). All sequenced insertions were scarless, but eight positive F1s from two cdh1::eGFP F0s had a single nucleotide change in the middle of the eGFP sequence (missense mutation without loss of fluorescence), maybe due to error during PCR donor synthesis or during HDR.