Experiments involving animals were conducted at the University of São Paulo Medical School. The study protocols were sanctioned by the University’s Ethics Committee on Animal Use (CEUA), approval number 1675/2021. The zebrafish were housed in 3.5-liter aquariums provided by Altamar/Brasil, with each containing 15 adult fish. Environmental conditions were rigorously controlled: pH ~7.5, conductivity between 500-700 μS/cm, and water temperature maintained at 28°C ± 1. A photoperiod regimen of 14 hours light and 10 hours darkness was established. The zebrafish were sustained at the Central Animal Facility of the Medical School, USP, and received Gemma micro commercial feed (Skretting, a Nutreco company^®) three times daily, the feed size commensurate with the age of the fish, ranging from 75 to 300 microns.

To pinpoint the most accessible regions for sgRNA targeting, we performed in silico analysis using the RNAfold software (Lorenz et al., 2011). Inputting the cDNA sequence into the software, we opted for settings that would minimize Free Energy (MFE) and identify Partition Function while avoiding isolated base pairs. We visualized RNA secondary structure plots to locate 22–23 nucleotide protospacers with high accessibility, indicated by a low probability of base-pairing, within the Coding Sequence (CDS). We selected three distinct protospacers, each spaced at least 50 nucleotides apart throughout the transcript to avert overlap of guide RNAs (gRNAs) ( Table 1 ). This strategy was integral for the successful identification of prime target sites within the mRNA for precise and effective silencing. A guide was manufactured as a positive control for the tbxta gene as described by Kushawah G at al, 2020 (26). The protospacers were synthesized as single-stranded oligonucleotides by Thermo Fisher.

The pT3TS-RfxCas13d-HA plasmid, obtained from Addgene (Plasmid #141320, http://www.addgene.org/141320/), was propagated according to Addgene’s specifications. We cultured ampicillin-resistant bacteria on brain heart infusion (BHI) agar medium supplemented with 100 μg/ml ampicillin. Post a 24-hour incubation at 37°C, a single colony was propagated in liquid BHI medium for another 24 hours at 37°C. Following centrifugation (10000g for 30 seconds), we discarded the supernatant and resuspended the pellet in a solution1 containing 25 mM Tris-HCl, 50 mM glucose, and 10 mM EDTA, followed by the addition of solution 2 (0.2N NaOH and 1% SDS). After 5 minutes on ice incubation, we added a cold solution 3 (120 mL 5M Potassium Acetate, 23 mL acetic acid, and 57 mL MilliQ water), followed by another 5 minutes ice incubation. Post-centrifugation (12000g for 5 minutes), the supernatant was mixed with cold 100% ethanol and 3M sodium acetate and incubated at -20°C and centrifuged at 4°C for 30 minutes the next day. The resultant pellet was resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA).

Two micrograms of the plasmid were digested with BamHI restriction enzyme for 4 hours, and successful digestion was confirmed via agarose gel electrophoresis by using 1μL of the reaction applied to a 1.5% agarose gel. The digested sample was purified using a commercial kit (TIANquick mini purification Kit) and then quantified using nanodrop spectrophotometer. For in vitro transcription, we employed the T7 mMessage mMachine kit. The transcribed RNA was verified on an agarose gel, purified using the gene art precision kit (thermofisher), and quantified on nanodrop spectrophotometer. The primers for the sgRNAs were synthesized in the form of DNA templates, and in vitro transcription was performed using a commercial kit, as described in the protocol by Kushawah et al., 2020 (16).

After producing the sgRNAs and transcribing the plasmids, the samples were quantified using a Nanodrop spectrophotometer, and the injection mix was prepared with 300 ng/μL of plasmid RNA and 900 ng/μL of total sgRNA (300 ng of each sgRNA). The injection needles were prepared from borosilicate glass capillaries (Sutter Instrument, USA) using a micropipette puller (Sutter Instrument, USA). The needles were filled with 5 μL of the knockout, knockin, or knockdown mix using a microloader (Eppendorf). Next, the needle was placed in a holder connected to a pneumatic microinjection pump (Harvard Apparatus, USA).

Injections were performed at 6.3X magnification under a stereomicroscope (SMZ 745, Nikon, Germany). The injection volume was calibrated using a micrometer ruler (Bresser 1/10 mm), according to the formula for the volume of a sphere (V = 1/6πd^3), where a sphere diameter of 1 bar corresponds to an injection volume of 1 nL.

To initiate the injection, the embryos were placed in a Petri dish with an agarose mold (WPI, USA) to prevent them from slipping under the needle pressure. 1 nL of the respective mix was injected into the single-cell stage embryo.

Gene expression levels of cdh2, gh1, prop1, and pit1 were assessed by Real-Time PCR in zebrafish embryos at key developmental stages (24, 48, 72, and 96 hpf) we utilized 20 embryos per pool and performed 5 distinct pools. We utilized the endogenous gene ef1a as a normalization control. RNA was extracted following the Trizol method and stored at -80°C after elution in Milli-Q water. Total RNA was quantified with a NanoPhotometer P-300 (Implen, Munich, Germany), and cDNA was synthesized from 1000 ng of total RNA using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. Primers used are detailed in Table 2 . cDNA samples underwent PCR using SYBR™ Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and amplified in an AriaMx Real-Time PCR System (Agilent, Santa Clara, CA, USA). Gene expression was normalized against wild-type controls and the least variable endogenous gene. The relative quantification was calculated using the 2^-ΔΔCT method by Livak and Schmittgen (2001) (27) where ΔCT is the difference in expression between the target and endogenous control genes, and ΔΔCT is the ΔCT difference between the mutant and wild-type samples for the same developmental period. Each sample was processed in triplicate. The nucleotide sequences of the primer pairs used are described in Table 2 . The primers for the ef1a, pit1 (28), and gh1 (29) genes have been previously reported in the literature. The primers for amplifying the prop1 and cdh2 genes were designed in our laboratory.

The presence of cdh2 protein in wild-type and knockdown samples was determined by Western blot. We collected pools of 30 embryos at 24 hpf and 96 hpf, and protein extraction was carried out. Briefly, samples were lysed in 100 μL of RIPA buffer (ThermoFisher Scientific, USA) and protease inhibitor (Sigma, St. Louis, USA). Each sample was quantified using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Massachusetts, USA). Equal amounts of protein (50 μg) dissolved in SDS sample buffer were loaded onto 12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gel wells and subjected to electrophoresis in a mini-gel tank (Bio-Rad, Brazil) for 2 hours at 120V. Following electrophoresis, proteins separated in the gel were transferred to a nitrocellulose membrane. The membranes were then blocked with 5% skim milk (Molico, Brazil) diluted in TBST (1M Tris-HCL, 3M NaCl, and 0.2% Tween 20) for 2 hours. After blocking, the membrane was incubated in a solution of 5% skim milk and PBST with primary antibody Anti CDH2 (ABCAM) diluted at 1:500 and/or Anti-Actin (SIGMA) diluted at 1:5000 and incubated at 4°C overnight with agitation. Three quick washes with PBS were performed after incubation, followed by three 10-minute washes with PBST. Then, the membrane was incubated with secondary anti-Rabbit antibody conjugated with HRP diluted at a concentration of 1:5000 in PBST for 2 hours at 4°C. The antigen-antibody immunocomplex was visualized with SuperSignal West Pico Plus chemiluminescent substrate (Thermo Scientific, Rockford, USA). Band intensities were visualized using an imaging capture system (UVITEC, Cambridge, UK).

The animals were examined under a stereomicroscope (Nikon SMZ800) at 48 hours post-fertilization, and their morphology was analyzed.

To compare two groups, both parametric and non-parametric tests were applied based on data distribution. For normally distributed data, an unpaired Student’s t-test was used. For non-normally distributed data, a Mann-Whitney U test was applied. Statistical significance was set at p < 0.05. All statistical analyses were performed using GraphPad Prism software, with results presented as mean ± standard error of the mean (SEM).

The efficacy of the technique was evaluated by observing the phenotypes of the animals and measuring the expression levels of the cdh2 gene in cdh2 knockdown animals. For greater reliability of the technique and better visualization of its efficiency, we performed a positive control with guides for the tbxta gene (no tail), where the animal is formed without the tail, these guides are provided in the pipeline by Kushawah G, et al., 2020 (16). Stereomicroscopic analysis revealed that animals injected with sgRNA for the tbxta gene (a positive control) exhibited tail shortening, which confirmed the effectiveness of the method. Conversely, animals injected with sgRNA targeting the cdh2 gene mRNA displayed developmental deficiencies like head malformation, and developmental delay. These phenotypes, although milder, were similar to those observed in cdh2 knockout animals (10) ( Figure 1 ).

In addition to the phenotypic analysis, the expression of cdh2 in knockdown animals was investigated to validate the efficacy of the technique in reducing gene expression. A 75% decrease in cdh2 gene expression was observed at 24 hpf ( Figure 2A ) in comparison to wild-type (WT) animals. From 48 to 96 hpf, there was a progressive restoration of cdh2 expression in knockdown animals ( Figure 2C ), as anticipated for the knockdown approach, whereas in WT animals, gene expression remained stable until 96 hpf ( Figure 2B ).

Western blot analysis supported the gene expression results obtained by RT-qPCR, confirming the reduction of the cdh2 protein in knockdown animals. At 24 hpf, the protein expression was reduced by 64% compared to WT animals, as shown in Figures 3A, B . Notably, at 96 hpf, protein expression in knockdown animals appeared to be more substantial than in WT animals, suggesting a compensatory overexpression following the initial suppression. These results corroborate the effectiveness of the knockdown approach in significantly reducing the expression of the cdh2 protein initially, followed by a recovery of protein production over time. This recovery suggests that the knockdown not only transiently blocked cdh2 gene expression but also permitted a full restoration of protein production, elucidating the role of the cdh2 gene and the n-cadherin protein in development and validating the reversibility of the knockdown method.

We examined the effect of cdh2 suppression on embryonic hormone production by analyzing the gene expression of markers such as gh1 (growth hormone), prop1 (PROP Paired-Like Homeobox 1), and pit1 (POU class 1 homeobox 1) through RT-qPCR. The temporal expression pattern of gh1 was inversely related to WT animals, where expression in WT began higher and decreased over time, whereas in knockdown animals, it started lower and increased ( Figures 2D, E ). The production of gh1 was statistically lower at 48 hpf in knockdown animals compared to WT ( Figure 2F ). At 72 hpf, the trend was toward a decrease compared to WT ( Figure 2G ), and at 96 hpf, when cdh2 production paralleled WT, gh1 expression recovered to levels comparable to WT ( Figures 2D, E ).

Regarding the markers prop1 and pit1, analyses were initiated at 24 hpf and extended up to 96 hpf, with evaluations conducted every 24 hours. In Figure 2H , we can observe the temporal expression of pit1 in the cdh2 knockdown group, showing a trend toward decreased expression at 48 hpf, although it was not statistically different from WT animals ( Figure 2I ). The expression of prop1 remained constant in cdh2 knockdown animals from 24 to 96 hpf ( Figure 2J ) and was equivalent to WT at 48 hpf ( Figure 2K ) when the gh1 was decreased.