Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish

Summary

Here, we present a protocol to label proteins with a fluorescence protein tag in zebrafish larvae, a newly developed and modified in vivo system especially useful for visualizing low-abundance proteins in zebrafish.

Abstract

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in vivo imaging due to its transparency during the early developmental stage. Here, we provide a detailed protocol for performing high-efficiency fluorescence gene KI, rapid screening for KI founders, and low-abundance protein tracing in zebrafish larvae, which will lay a critical foundation for subsequent physio-pathological studies in zebrafish. The current protocol includes complete steps for the sgRNA design for the gene of interest, sgRNA in vitro transcription, Cas9 mRNA in vitro transcription, in vivo sgRNA screen for the one with the highest efficiency, donor plasmid design and construction, microinjection in zebrafish larvae, KI founder screen and zebrafish live imaging. Critical steps, troubleshooting tips, quality control methods, and advantages and applications of this protocol are included and discussed. This protocol assures quick and accurate results at a low cost and has been validated by multiple trials.

Introduction

As a well-accepted model organism, zebrafish (Danio rerio) is widely used in scientific research on development, regeneration, tumorigenesis, infection and immunity, etc^1,2,3,4,5. Zebrafish are especially suitable for in vivo live imaging when treated with PTU (1-phenyl 2-thiourea), a toxic chemical compound that inhibits melanogenesis and makes zebrafish larvae transparent for a relatively long period of time⁶. Genetically modified transparent zebrafish strains, such as the casper⁷ and the crystal⁸, are also available, allowing for live visualization and lineage tracing till adulthood. Besides these efforts to overcome pigmentation for better live visualization in adult fish, our research also focuses on developing powerful gene editing strategies to make fluorescent protein tagging more efficient and less artificial.

CRISPR/Cas9-mediated microhomology-mediated end joining (MMEJ)-based KI technology is now widely applied in genetic modifications in multiple organisms, including zebrafish, due to its relatively high KI efficiency^9,10,11,12. To achieve successful in vivo imaging, two challenges need to be addressed. One is the indels or scars introduced by the process of recombination; the other one is that some target genes are endogenously expressed at a low level, leading to weak signals of the co-expressed fluorescence tag and the failure of in vivo detection. Aiming for efficient and accurate protein tagging and minimizing the interference of endogenous gene expression levels, a recent work reported an optimized strategy combining a fluorescence-signal-amplifying "VH" tagging method and a refined germline screen workflow¹³, which is more suitable for fluorescence labeling low expressed genes and more efficient than previous methods by optimizing the donor backbone and combining polymerase chain reaction (PCR) and fluorescence verifications to implement germline screen at the lowest cost of time and effort. Here, we present a detailed step-by-step protocol for the reported method, including steps of embryo preparation and zebrafish husbandry, sgRNA design, in vitro transcription of sgRNAs, in vitro transcription of Cas9 mRNA, zebrafish embryo microinjection, in vivo screen for sgRNAs with the highest efficiency, donor plasmid design and construction, KI zebrafish genotyping, KI germline screen, and KI zebrafish imaging and phenotyping. This all-in-one protocol, providing a state-of-the-art fluorescence labeling method, would no doubt promote studies using zebrafish in various scientific fields and benefit readers from different academic backgrounds.

Protocol

All zebrafish husbandry and experiments were reviewed and approved by the Laboratory Animal Management and Ethics Committee of Xiamen University and were in strict accordance with good animal practice as defined by Xiamen University Laboratory Animal Center. The study complied with all relevant ethical regulations for animal use.

NOTE: All adult and larval zebrafish were maintained following standard protocols at 28.5 °C with a 13/11 h light/dark cycle in the zebrafish aquarium system of Xiamen University. Embryos were kept in the embryo buffer (Table 1) in a controlled incubator (temperature, 28 ± 0.5 °C; light, 13 h light/11 h dark cycle). Feeding and general monitoring of all zebrafish were performed twice daily (9 A.M. and 4 P.M.). The wild-type (WT) strain used in this research was Tübingen (Tü).

1. Choosing genes of interest and designing the guide RNA

1. Know the information of the gene of interest. Search the database (https://zfin.org/) and make sure the number of gene variants is known. Decide which variant to manipulate and download target gene information from Ensembl (https://www.ensembl.org/), including genome sequence, cDNA sequence, and coding sequence (CDS). Mark exons, introns, UTRs, start codons, and stop codons in the sequences.
2. Choose no more than 200 bp of CDS upstream of the stop codon (TAA, TAG, or TGA) of the target gene as the sgRNA target region.
   NOTE: It is better to make sure this region has no single nucleotide polymorphisms (SNPs) by DNA sequencing.
3. Use the CRISPRscan website (https://www.crisprscan.org/) to design candidate sgRNAs within the chosen target region and predict sgRNA efficiencies. 
   1. Input the DNA sequence into the design frame.
   2. Select the corresponding information, including species, max mismatch(es) in off-targets, endonuclease, and promoter. In this case, choose Zebrafish- Danio rerio, No mismatch, Cas9-NGG, and in vitro T7 promoter.
   3. Click Get sgRNAs to obtain some sgRNAs. Select 5-7 sgRNAs with a high CRISPRscan score and a low off-target score for the in vivo screen.
4. Design PCR primers flanking the sgRNA target sites whose product is about 500 bp for subsequent PCR amplification, enzymatic digestion test, and sequencing to verify the knockout efficiency after sgRNA/Cas9 microinjection. Select a sequence of approximately 20 bp in upstream and downstream regions and ensure its GC content is about 50% and the Tm value ranges from 50-55°C.
   NOTE: Design two upstream primers and two downstream primers if there is a need to do combinations.

2. Preparation of sgRNAs

NOTE: The common sgRNA scaffold sequence (Figure 1) has been cloned into a plasmid (Table of Materials).

1. PCR amplify the T7 promoter-sgRNA conjugate sequence using primers and PCR mix shown in Table 2 and Table 3, respectively. Run the PCR program in Table 4. Run gel electrophoresis (Agarose gel, 1.2% agarose in TAE buffer; TAE buffer [Table 1]; 200 V; about 5 min) with 2 µL of the PCR products to make sure that the products are correct (the MW is about 150 bp).
2. Purify the PCR products using a purification kit (Table of Materials) or other commercially available kits following the manufacturer's instructions.
   NOTE: From now on, RNase-free reagents and materials should be used, and procedures should be conducted with special caution to avoid RNase contamination (see Discussion).
   1. Add the binding buffer to the PCR products at a 5:1 volume ratio and mix. Apply the mixture to the column placed in a 2 mL collection tube. Centrifuge at 17,900 × g for 1 min at 25 °C. Discard the flow-through and place the column back into the same collection tube.
   2. Add 750 µL of the wash buffer to the column and centrifuge at 17,900 × g for 1 min at 25 °C. Discard the flow-through and place the column back into the same collection tube. Centrifuge at 17,900 × g for 2 min at 25 °C. Place the column in a clean 1.5 mL tube.
   3. Add 20 µL of nuclease-free water to the column, centrifuge at 17,900 × g for 2 min at 25 °C, to elute the DNA and measure the concentration using a spectrophotometer.
3. Use the T7 RNA Production System in Table of Materials or equivalent kits to in vitro transcribe 250 ng of the purified DNA. RNase-free operations and conditions are required.
   1. Prepare the reaction in PCR tubes (RNase-free) according to Table 3, and mix gently. Incubate at 37 °C in a metal bath for 2 h.
   2. Add 0.5 µL of DNase I (RNase-free), and mix gently. Incubate at 37 °C in a metal bath for 15-20 min.
   3. Ethanol precipitation
      1. Add RNase-free water to each reaction to make the final volume reach 20 µL, and transfer the samples to 1.5 mL tubes.
      2. Add 50 µL of 100% ethanol (2.5V, RNase-free), mix well, put the mix on ice for 10 min, and centrifuge at 21,000 × g for 15 min at 4 °C.
      3. Discard the supernatant, wash the pellet with 500 µL of 75% ethanol (RNase-free), and centrifuge at 21,000 × g for 5 min at 4 °C.
      4. Discard the supernatant, dry the pellet at 45 °C in a metal bath, and dissolve with 40 µL of RNase-free water.
         NOTE: For KI, sgRNAs need to be LiCl-precipitated (see below), but for KO, it is not necessary.
   4. Run agarose gels (Agarose gel: 1.2% agarose in TAE buffer; TAE buffer [Table 1]; 200 V; about 5 min) with 1-2 µL of the RNA to estimate the quality and then measure the concentration using a spectrophotometer.
      NOTE: sgRNAs could be stored at -20 °Cor -80 °Cfor further use.

3. Preparation of Cas9 mRNA

1. Perform enzymatic digestion of the Cas9 plasmid (Table of Materials) for more than 1.5 h using XbaI according to the manufacturer's instructions (for the reaction mix, see Table 3). Test by gel electrophoresis (Agarose gel: 1.2% agarose in TAE buffer; TAE buffer [Table 1]; 200 V; about 10 min) with 1 µL to check that the digestion is thorough.
2. Purify the products in the same way as sgRNA PCR products (steps 2.2.1-2.2.3). From here on, RNase-free materials and operations are required.
3. Use the T3 transcription kit (Table of Materials) or equivalent kits for in vitro transcription of Cas9 mRNA.
   1. Prepare the reaction in RNase-free PCR tubes according to Table 3 and mix gently. Incubate at 37 °C for more than 2.5 h.
   2. Add 1 µL of RNase-free Turbo DNase and incubate at 37 °C for 15-20 min.
   3. LiCl precipitation
      1. Transfer the samples to 1.5 mL tubes, add 30 µL of LiCl (1.5 V), and mix gently.
      2. Put the mix at -20 °C for about 30 min and then centrifuge at 21,000 × g for 30 min at 4 °C.
      3. Discard the supernatant, wash the pellets with 500 µL of 75% ethanol (RNase-free), and centrifuge at 21,000 × g for 5 min at 4 °C.
      4. Discard the supernatant, dry the pellet at 45 °C, and dissolve it in 40 µL of RNase-free water.
4. Measure the concentration of the Cas9 mRNA using a spectrophotometer. Run agarose gels (Agarose gel: 1.2% agarose in TAE buffer; TAE buffer [Table 1]; 200 V; about 10 min) with 1-2 µL of the RNA to check the concentration and quality.
   NOTE: An OD 260/280 should be above 2.0, and the concentration should be around or higher than 1000 ng/µL. Cas9 mRNA could be stored at -20 °Cor -80 °Cfor further use.

4. Preparation for the microinjection

1. Prepare the zebrafish: The night before microinjection, place a healthy female zebrafish and two healthy male zebrafish in a mating tank, using a divider to separate the males and females to prepare them for mating.
2. Prepare 4% agarose, lysis buffer, and embryo buffer (pH 8.0) (Table 1).
3. Prepare capillary glass needles for the microinjection (Table of Materials).
4. Prepare microinjection plates: Pour 35 mL of the melted 4% agarose into a 10 cm Petri dish and gently lay a plastic mold to create troughs of a wedge shape. Avoid air bubbles by gently tapping the mold. After it solidifies, add some double distilled water (ddw) to it and place the plate at 4 °C for later use.

5. sgRNA efficiency test through microinjection

1. On the day of microinjection, prepare the microinjection mix as listed in Table 5 on ice under an RNase-free environment.
2. Load the microinjection solution into the needle using an RNase-free pipette tip (Table of Materials). Be careful not to bring bubbles.
3. Place the loaded needle in a micromanipulator attached to a microinjector. Cut the tip of the injection needle under the microscope with the pointed tweezers, gently pinching at the tip of the needle to break it. Then, adjust the injection pressure until the needle consistently ejects a 1-nL droplet, which is quantified by using a stage micrometer and a capillary.
4. Remove the divider and allow the fish to breed for about 6 min. Collect and transfer the 1-cell-stage embryos into a Petri dish containing embryo buffer.
   1. Examine the health of the embryos under the light microscope and remove any unfertilized eggs or debris. Set aside 20 healthy embryos as uninjected controls in a separate Petri dish and label.
5. Transfer and gently line up healthy 1-cell-stage embryos onto the grooves of the microinjection plate warmed to room temperature (RT) using a small brush. Remove excess buffer.
6. Perform all subsequent operations under a stereo microscope. Use the needle tip to gently adjust the position of the embryo so that the animal pole faces the needle. Inject 1 nL of the mixed solution into the animal pole.
7. Approximately the injection of 100 embryos is sufficient for one sgRNA. After injection, gently rinse the embryos into a six-well plate with embryo buffer. Put the dish into a 28.5 °C constant temperature incubator. At around 10 h post fertilization (hpf), examine the embryonic development under a stereo microscope and remove dead embryos.
8. At 24 hpf, collect embryos in 1.5 mL tubes for genotyping (3 tubes per sgRNA and 5 embryos per tube). Discard extra water carefully with a pipette, add 100 µL of lysis buffer containing Proteinase K (at a final concentration of 0.5 mg/mL) into each tube, and incubate at 56 °C for 1 h and then 95 °C for 15 min.
9. Add 200 µL of ethanol (2V), mix well, and centrifuge at 19,000 × g for 10 min at RT.
10. Discard the supernatant, wash the pellet with 500 µL of 70% ethanol, and centrifuge at 19,000 × g for 3 min at RT.
11. Discard the supernatant, dry the pellet at RT, and dissolve it with 50 µL of ddw. Then, use 1 µL of the extracts to prepare the reaction mix for the PCR (Table 3) (for PCR primers, see step 1.4).
12. Run agarose gels (Agarose gel: 1.2% agarose in TAE buffer; TAE buffer [Table 1]; 200 V; about 10 min) with 2 µL of the PCR products to confirm that the PCR is successful, and then perform enzymatic digestions (Table 3) to determine the efficiency of the sgRNAs. Gently mix the enzymatic digestion mix. Incubate at the temperature for the period of time suggested by the manufacturer.
13. Run agarose gels for the digested products (Agarose gel: 1.2% agarose in TAE buffer; TAE buffer [Table 1]; 200 V; about 10 min) with the not-digested PCR products as controls to confirm whether the digestion occurs.
    NOTE: The Cas9 cleavage site is about 3 bp upstream of the PAM, so the restriction enzyme recognition site is best for covering 6 bp upstream of PAM. If the sgRNA efficiency is high, cutting and mis-repair will lead to indels, so there is a high possibility that the restriction enzyme can no longer recognize the cutting site. If the sgRNA is inefficient and the restriction enzyme recognition site is intact, the enzymatic digestion can occur normally. Through agarose gel electrophoresis, one can distinguish whether enzymatic digestion has occurred.
14. If there is no suitable restriction enzyme recognition site, sequence the PCR products and analyze the sgRNA efficiency by TIDE (https://tide.nki.nl/). Remember to do PCR and also sequence WT genomic DNA as a control.
    1. Open the website and click Start TIDE. Input the guide sequence of sgRNA in the frame under Guide sequence.
    2. Click Browse under Control Sample Chromatogram (.ab1 or .scf) to input the sequencing result for the WT control and click Browse under Test Sample Chromatogram (.ab1 or .scf) to input the sequencing result for the edited sample.
    3. Click Update View. The website will give a percentage of the difference between the two sequences. A larger percentage indicates a higher sgRNA efficiency. A sgRNA with a knockout efficiency above 80% is suitable for subsequent KI procedures.

6. Donor plasmid construction for fluorescent protein tagging

1. Design sequences that would be first cloned into the plasmid and then further inserted into the fish genome as Figure 2: a 25-bp sequence upstream of the predicted Cas9 cleavage site is designed as the left homology arm, and a 25-bp sequence downstream of the predicted Cas9 cleavage site is the right homology arm. Introduce CDS between the sgRNA target site and the stop codon into the donor to maintain the integrity of the endogenous gene.
   NOTE: The 5' of the complemented CDS (element 4 in Figure 2) needs to be synonymously mutated to avoid secondary cleavage of the recombinant genome by the sgRNA/Cas9 complex.
2. Use ligation-independent cloning (LIC)¹⁴ using exonuclease III (Table of Materials) for the donor plasmid construction. Other methods, such as the Gibson assembly, are also feasible. Cloning primers are illustrated in Figure 2. Elements "1", "2", "3" and "4" are carried by the primers:
   1. Design Primer F1, which is composed of an overlapping sequence with the vector, element "1" and a part of element "2".
   2. Design Primer R2, which is composed of a part of element "1", element "2" and element "3".
   3. Design Primer F2, which is composed of elements "3", "4", and the 5' sequence of the CDS following the sgRNA target site.
   4. Design Primer R1, which is composed of the 3' sequence of the CDS of the gene and an overlapping sequence with the vector.
   5. Use zebrafish cDNA as the template and do PCRs using the primers.
      NOTE: Recommended overlap between primers or between the primer and the vector is 15-20 bp. These primers are target-gene specific, and an example of the primers for cx39.9 and cx44.1 is given in Table 2. This design was successfully applied as previously described¹³. Detailed plasmid maps are provided in Supplementary File 1 and Supplementary File 2. The designed DNA and protein sequences of cx39.9 and cx44.1 are provided in Supplementary File 3.
   6. Purify the PCR products in the same way as sgRNA PCR products (steps 2.2.1-2.2.3).
3. Digest the donor vector (Figure 3 and Table 6) by proper restriction enzymes. In this example, the enzymes are PciI and Acc65I (Table of Materials). Conduct the according to the manufacturer's instructions (Table 3). Purify the digested donor vector in the same way as sgRNA PCR products (steps 2.2.1-2.2.3).
4. Quantify the purified PCR fragments and digested donor vector by DNA gel electrophoresis (DNA gel: 1.2% agarose in TAE buffer; TAE buffer [Table 1]; 200 V; about 10 min). Prepare the reaction mix for LIC according to Table 3.
   NOTE: All tubes and components of the reaction should be placed on ice, and the reaction and all manipulations should be performed on ice. Add 1 µL of Exo III (20 units) to the reaction as the last component and then immediately pipette up and down to mix all components well. Remember to include one reaction mix without PCR fragments as a control to show the potential of self-annealing of the digested vector.
5. Immediately place the reaction mix on ice and incubate for 60 min.
6. Add 1 µL of 0.5 M EDTA (pH 8.0) to stop the reaction, pipette up and down to mix well, and then incubate at 60 °C for 5 min and then on ice for 5 min.
7. Transform competent cells, such as the DH5α, using the LIC products and grow the bacteria on LB plates (Table of Materials and Table 1) with proper antibiotics (Ampicillin in this case) for 16 h.
8. Ensure that there are orders of magnitude differences between the number of colonies on the PCR-fragment-containing plates and that on the control plate. Pick out single colonies from the LIC plates for enzymatic digestion analysis and DNA sequencing.
9. Extract and purify correct donor plasmids with a purification kit in the same way as sgRNA PCR products (steps 2.2.1-2.2.3). Ensure that the final concentration is above 300 ng/µL.

7. Microinjection of zebrafish embryos for gene KI

1. Perform all microinjection manipulations described above, except preparing the microinjection solution according to Table 7.

8. Screening and identification of fluorescent protein-labeled progeny

1. Observe fluorescence under a stereo fluorescence microscope 12-48 h after microinjection.
2. Apply a newly established screen method¹³. If obvious fluorescence is observed, pick out the fluorescent larvae and rear them. If only a trace of fluorescence or even no fluorescence is observed, rear all injected larvae.
3. At 1 month of age, anesthetize microinjected zebrafish with 0.02% tricaine (Table 1). For each zebrafish, collect a small piece of the caudal fin into a labeled PCR tube and keep the corresponding anesthetized zebrafish in one well of a six-well plate containing UV-sterilized filtered water (pH 7.2) with the same label.
4. Use the alkaline lysis method (lysis buffer in Table 1)¹⁵ for genomic DNA extraction, followed by the 5'-junction PCR. Design the forward primer upstream of the left homology arm and the reverse primer on the insert (Figure 4). The size of the PCR product is around 500 bp. For examples of the primers, see Table 2.
   NOTE: For the fluorescent zebrafish, this PCR is used to verify the accuracy of the KI, so the PCR product is then subjected to sequencing. For zebrafish without fluorescence, this PCR is used to verify whether KI occurs. Pick out the PCR-positive fish and raise them to adulthood as F₀.
5. Examine F₁ embryos obtained from mating the screened F₀ with WT for GFP expression patterns and genotyped by junction PCR.
   NOTE: If target insertion occurs in F₁ (whether perfect or not), the corresponding F₀ is defined as heritable F₀, the PCR product of which is then sequenced. If the insertion is accurate, the corresponding F₀ is defined as the desired F₀.
6. When the desired F₀ is obtained, use the progeny for imaging to observe and track the target protein.

9. Imaging and data processing

1. Determine the developmental stage to be imaged. Prepare the fish in advance. Add diluted 1-phenyl 2-thiourea (PTU; 0.003%, Table of Materials) to the fish to remove the pigments if necessary.
   NOTE: PTU would be less effective if applied after 24 hpf. In this example, PTU was added to the embryo buffer before 24 hpf and kept in the buffer till 48 hpf when the zebrafish larvae were processed for imaging.
2. Add 2 mL of melted 4% agarose (Table 1) to a 35-mm dish and leave it aside to solidify. Transfer the fish into the embryo buffer containing 0.02% tricaine (Table 1).
3. Dilute 200 µL of melted 4% agarose in 1 mL of ddw. Transfer the anesthetized fish into the diluted agarose using a 1-mL pipette when the agarose is cooling down. Gently mix and transfer the fish-mounting agarose into the dish containing solidified 4% agarose gel prepared in step 9.2.
4. Position the fish so that the part of interest is on the top.
5. Use an upright confocal fluorescence microscope for imaging and data processing following the manufacturer's instructions (Table of Materials).

Results

Gap junctions consisting of polymeric proteins called connexins are essential for the exchange of low-molecular-weight metabolites and ions between contacting cells. The fluorescent protein-tagging method described above was used to label connexin 39.9 (Figure 5A) and connexin 44.1 (Figure 5B), respectively. Correct KI was verified by junction PCRs (Figure 6). In agreement with published data¹⁶

Discussion

To conduct this experiment smoothly and successfully, one should pay more attention to some important aspects. The optimal zebrafish developmental stage for injection should be the one-cell stage. Completing injection within this stage can ensure that all cells produced through subsequent division contain the necessary components for KI, thereby increasing the probability of simultaneous KI occurrence in all cells and improving the overall success rate of KI. Lowering the ambient temperature would slow down zebrafish emb...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1000 µL  tips	Any brand	N/A	For all manipulations
1.5 mL tubes	Any brand	N/A	For storage and centrifugation of various solutions
1.5 mL tubes (RNase-free)	Any brand	N/A	For storage and centrifugation of RNase-free solutions
10 µL tips	Any brand	N/A	For all manipulations
10 µL tips (RNase-free)	Any brand	N/A	For RNase-free manipulations
1000 µL  tips (RNase-free)	Any brand	N/A	For RNase-free manipulations
1-phenyl 2-thiourea (PTU)	Sigma	P7629	To inhibit melanogenesis and keep the larvae transparent
200 µL tips	Any brand	N/A	For all manipulations
200 µL tips (RNase-free)	Any brand	N/A	For RNase-free manipulations
2x Taq Plus Master Mix II (Dye Plus)	Vazyme	P213-03	PCR for sgRNA efficiency tests
35-mm dishes	Corning	430165	For imaging
6-well plates	Any brand	N/A	For rearing zebrafish individually and temporarily
Acc65I	NEB	R0599S	For enzymatic digestion
Acetic acid	Sigma	695092	For TAE buffer preparation
Agar	Biofroxx	8211GR500	For LB plates
Ampicillin, sodium salt	Sangon	A610028	For LB plates
CaCl2	Sigma	C5080	For embryo buffer preparation
Capillary tubes	Harvard Apparatus	30-0016	For geneating needles for injection
Capillary tubes	Any brand	ID (inside diameter) = 0.1 mm	For quantify a 1-nL droplet
CRISPRscan	CRISPRscan	http://www.crisprscan.org/	For sgRNA design
DH5α	TIANGEN	CB101	For transformation
EDTA	Sigma	E6758	For lysis buffer and TAE buffer preparation
Ensembl	Ensembl	https://asia.ensembl.org/Danio_rerio/Info/Index	To find gene information
Ethanol	HUSHI	64-17-5	For DNA purification and extraction
Exonuclease III	Takara	2170A	LIC
Gas-powered micro-injector	Warner Instruments	PLI-100A	For microinjection
Gel electrophoresis system	WIX	WIX-EP3000	For gel electrophoresis
Gloves	Any brand	N/A	For all manipulations
Glucose	sgdbio	10010518	For LB plates
Glycerol	Sangon	A501745	For LB plates
gRNA-pMD19-T	China Zebrafish Resource Center (CZRC)	CZP3	sgRNA plasmid containing sgRNA scaffold
KCl	Sigma	P5405	For embryo buffer preparation
Lithium chloride precipitation solution	Thermofisher	AM9480	Precipitation of sgRNAs, RNase-free
Low melting point agarose	Sangon	A600015	For preparation of 4% agarose gels
Magnesium sulfate	sgdbio	20025118	For LB plates
Metal bath	Any brand	N/A	For constant temperature incubation
Methylene blue	Sigma	M9140	For embryo buffer preparation
Microinection mold	Homemade	N/A	For generating grooves to hold embryos during microinjection
Microloader, tip for filling Femtotips and other glass microcapillaries, sterile, 0.5 – 20 µL, 100 mm, light gray, 192 pcs. (2 racks × 96 pcs.)	Eppendorf	5242956003	To Load the microinjection solution into the injection needle, RNase-free
Micropipette puller	Sutter Instrument	P-97	For generating needles for injection
Micropipettes	Eppendorf	N/A	For all manipulations
Microwave	Any brand	N/A	For casting injection plate and agarose gels
MinElute PCR Purification Kit (50)	QIAGEN	28004	To purify PCR products and digestion products, RNase-free
mMESSAGE mMACHINE T3 Transcription Kit	Invitrogen	AM1348	In-vitro transcription of Cas9 mRNA, RNase-free
N2	Any brand	N/A	For microinjection
NaCl	Sigma	S5886	For embryo buffer and lysis buffer preparation
NaHCO3	Sigma	S5761	For embryo buffer preparation
NaOH	Sangon	A100173	For TAE buffer preparation
Nuclease-free water	Thermofisher	R0581	RNA related manipulations, RNase-free
PciI	NEB	R0655S	For enzymatic digestion
PCR machine	Any brand	N/A	PCR amplification
PCR strip tubes	Any brand	N/A	For PCR
PCR tubes	FEITONGKANG	blp-200	For PCR
Petri dish	Any brand	N/A	For rearing zebrafish larvae etc.
Phenol red	Sigma	P0290	For microinjection, RNase-free
Pipette holder	Warner Instruments	PLI-PH1	For microinjection
PrimeStar (Premix)	Takara	DR040A	For molecular cloning
Proteinase K	Merck	539480	For genomic DNA extraction
pT3TS (T3:zCas9-UTRglobin)	China Zebrafish Resource Center (CZRC)	CZP11	Cas9 plasmid template
Refrigerated centrifuge	Any brand	N/A	For RNA related centrifugation
Scale	Sartorius	BCE124-1CCN	For casting injection plate and agarose gels
Scissors	Any brand	N/A	For clipping adult fish tails
SDS	Sangon	A600485	For lysis buffer preparation
Small brushes	Any brand	N/A	For gently moving embryos on microinjection plates
Spectrophotometer	Thermofisher	NanoDrop 2000	For DNA/RNA concentration measurements
Stage micrometer	Any brand	DIV (Division) = 0.01 mm	For quantify a 1-nL droplet
Stereo fluorescence microscope	Olympus	SZX16	For observation of gene expression
Stereo microscope	Motic	SMZ-168	For observation and microinjection
T7 RiboMAX Express Large Scale RNA Production System	Promega	P1320	In-vitro transcription of sgRNAs, RNase-free
Temperature-controlled incubator	Any brand	N/A	For embryo incubation
TIDE	TIDE	https://tide.nki.nl/	For sgRNA efficiency analysis
Tricaine methanesulfonate	Sigma	A5040	For tricaine solution preparation
Tris	Sangon	A600194	For tricaine solution, lysis buffer and TAE buffer preparation
Tryptone	OXOID	LP0042	For LB plates
Tweezers	Any brand	N/A	For cutting injection needles
XbaI	NEB	R0145S	For enzymatic digestion
Yeast extract	OXOID	LP0021	For LB plates
Zebrafish aquarium system	ESEN	ESEN-AW-S1	For rearing adult fish
Zebrafish mating tanks (with a divider)	Any brand	N/A	For mating of the fish
Zeiss LSM 900+Airyscan2	ZEISS	Zeiss LSM900	For confocal imaging of the fluorescence-labeled fish and data analysis
ZFIN website	ZFIN	http://zfin.org/	To find gene information