Efficient PAM-Less Base Editing for Zebrafish Modeling of Human Genetic Disease with zSpRY-ABE8e

Podsumowanie

This protocol describes how to perform efficient adenine base editing without PAM limitation to construct a precise zebrafish disease model using zSpRY-ABE8e.

Streszczenie

CRISPR/Cas9 technology has increased the value of zebrafish for modeling human genetic diseases, studying disease pathogenesis, and drug screening, but protospacer adjacent motif (PAM) limitations are a major obstacle to creating accurate animal models of human genetic disorders caused by single-nucleotide variants (SNVs). Until now, some SpCas9 variants with broad PAM compatibility have shown efficiency in zebrafish. The application of the optimized SpRY-mediated adenine base editor (ABE), zSpRY-ABE8e, and synthetically modified gRNA in zebrafish has enabled efficient adenine-guanine base conversion without PAM restriction. Described here is a protocol for efficient adenine base editing without PAM limitation in zebrafish using zSpRY-ABE8e. By injecting a mixture of zSpRY-ABE8e mRNA and synthetically modified gRNA into zebrafish embryos, a zebrafish disease model was constructed with a precise mutation that simulated a pathogenic site of the TSR2 ribosome maturation factor (tsr2). This method provides a valuable tool for the establishment of accurate disease models for studying disease mechanisms and treatments.

Wprowadzenie

Single-nucleotide variants (SNVs) that cause missense or nonsense mutations are the most common source of mutations in the human genome¹. To determine whether a particular SNV is pathogenic, and to shed light on its pathogenesis, precise animal models are required². Zebrafish are good human disease models, exhibiting a high degree of physiological and genetic homology with humans, a short developmental cycle, and strong reproductive ability, which is advantageous for research into pathogenic characteristics and mechanisms, as well as drug screening³.

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has been widely applied in the genome editing of various species, including zebrafish⁴. With gRNA guidance, the CRISPR/Cas9 system can generate DNA double-stranded breaks (DSBs) at the target site, which then allows single-base substitution through recombination of the target site with donor DNA templates via the homology-directed repair (HDR) pathway. However, the efficiency of this base replacement method is quite low as the cellular DNA repair process is mainly carried out by the non-homologous end-joining (NHEJ) pathway, which is usually accompanied by insertion and deletion (indel) mutations⁵. Fortunately, CRISPR/Cas9-based single-base editing technology significantly alleviates this problem by using base editors, which enable more efficient single-base editing without inducing DSBs. Two major classes of base editors, adenine base editors (ABEs) and cytosine base editors (CBEs), have been developed to implement base substitution editing for A·T to G·C and C·G to T·A, respectively^{6,7,8,9,10,11}. These four types of base substitutions cover 30% of human pathogenic variants¹². Both classes of base editors, including PmCDA1, BE system, CBE4max, ABE7.10, and ABE8e, have been reported to work in zebrafish, with BE4max and ABE8e especially reported to achieve high editing activity^{13,14,15,16,17,18,19}.

Cas9 proteins from different species, including Staphylococcus aureus, Streptococcus pyogenes, and S. canis, have been implemented in zebrafish gene editing, with the Streptococcus pyogenes Cas9 (SpCas9) being used most widely^20,21,22,23. However, SpCas9 can only recognize target sites with an NGG protospacer adjacent motif (PAM), which limits its editable range and can result in no suitable sequence being found near the pathogenic site of interest²⁴. To expand the target range, a variety of SpCas9 variants have been engineered to recognize different PAMs through directed evolution and structure-guided design. However, few variants are effective in animals, especially in zebrafish, which limits the application of zebrafish in SNV-related disease research^25,26,27,28. Recently, two variants of SpCas9, SpG and SpRY, with less stringent PAM restrictions (NGN for SpG and NNN for SpRY with a higher preference for NRN than NYN) have been reported to exhibit high editing activity in human cells and plants^29,30,31,32. Subsequently, SpG and SpRY, as well as a number of their mediated base editors, such as SpRY-mediated CBEs and SpRY-mediated ABEs, have also been reported to work in zebrafish, which will enhance the application of zebrafish models in the mechanistic study and drug screening of SNV-related diseases^18,33,34,35. Furthermore, i-Silence was proposed as an effective and accurate gene-knockout strategy through ABE-mediated start codon conversion from ATG to GTG or ACG³⁶. The combination of the i-Silence strategy and the SpRY-mediated base editor zSpRY-ABE8e provides a new method for disease modeling¹⁸. This protocol demonstrates how to perform gene editing using zSpRY-ABE8e in zebrafish to construct a tsr2 (M1V) model using the i-Silence strategy. The editing efficiency and phenotypes that appear in zebrafish models were assessed and analyzed.

Protokół

This study was conducted in strict compliance with the guidelines of the Care and Use Committee of the South China Normal University.

1. Preparing synthetically modified gRNA and zSpRY-ABE8e mRNA

1. Design the gRNA according to previous publications^37,38.
   1. Preferentially select NRN as the PAM sequence, as zSpRY-ABE8e shows a higher preference for NRN PAM than NYN PAM (where R is A or G, and Y is C or T)¹⁸. Ensure that the target adenine nucleotide is in the highly active editing window (third to ninth nucleotide along the protospacer).
2. Order EasyEdit sgRNA (EE gRNA) modified with 2′-O-methyl-3′-phosphorothioate (MS) at both ends (Figure 1).
   NOTE: The EE gRNA should be dissolved in RNase-free water and stored at −80 °C.
3. Linearize the zSpRY-ABE8e plasmid¹⁸.
   1. Linearize 6 µg of the plasmid with the XbaI enzyme (Table of Materials) in a metal bath at 37 °C for 3 h.
   2. Purify the linearized plasmid using the DNA purification kit (see Table of Materials) according to the manufacturer's instructions.
4. Transcribe the mRNA using the in vitro transcription kit (Table of Materials).
   1. Transcribe the zSpRY-ABE8e mRNA with the linearized plasmid as template according to the manufacturer's instructions.
      NOTE: All operations involving mRNA and gRNA require the use of RNase-free laboratory supplies.
5. Use the RNA purification kit (Table of Materials) to purify the mRNA according to the manufacturer's instructions.
   NOTE: Before eluting, the ethanol must be dried to avoid cytotoxicity, and the mRNA should be stored at −80 °C if not used immediately.

2. Preparing microinjection glass capillaries

1. Set up the micropipette puller system, and preheat for at least 30 min.
2. Run a ramp test to determine the heat value needed to melt the borosilicate glass capillaries (with 1 mm outer diameter and 0.58 mm inner diameter) according to the manufacturer's instructions.
3. Select a program, and click on the number on the keyboard to enter the parameter values. Set the following parameters to pull the glass capillaries: heat = ramp test value, pull = 50, velocity = 100, time = 50, and pressure = 30.
4. Install the capillary tube, ensuring it is in the groove. Press PULL START/STOP to run the program.
5. Preserve the pulled capillaries by inserting them into foam that contains gaps, keeping the needle suspended.

3. Microinjection of the zSpRY-ABE8e mRNA and EE gRNA mixture into zebrafish embryos

1. Set up breeding tanks the night before the injection. Insert a divider, place two females and one male AB strain zebrafish (3-18 months old) in each tank, and cover with the lid. Refer to previous publications for gender distinctions³⁹.
   NOTE: To obtain more embryos, select fish that have not been breeding for at least 1 week.
2. The next morning before the injection, thaw the zSpRY-ABE8e mRNA and EE gRNA on ice. Mix the mRNA and gRNA to final concentrations of 400 ng/µL and 200 ng/µL, respectively.
   1. Perform the entire procedure on ice using RNase-free tips and tubes, and handle with gloves.
3. Configure the pneumatic microinjector. Close the partial pressure valve of the air pump and open the main valve first, unscrew the gas cylinder, and adjust the pressure of the partial pressure valve to 0.2 MPa/29 psi.
   1. Turn on the pneumatic microinjector, set the mode to TIMER, and adjust the initial parameter pressure value to 20.0 psi and the TIMER value to 0.040 s.
4. Use fine forceps to carefully break the needles of the pulled glass capillaries under a stereomicroscope, making sure the needles are cut at an angle to facilitate the piercing of the chorion and the egg.
   NOTE: The break point of the needle needs to be in the right place and be strong enough to pierce the egg yet thin enough to minimize damage to the egg.
5. Add the mixture of zSpRY-ABE8e mRNA and gRNA to the tip of a glass capillary using a microloader pipette tip, avoiding bubbles in the capillary. Attach the needle to the micromanipulator, and configure the pressure and TIMER value of the injector to ensure 2 nL of mixture is produced per injection using a microcap.
6. Unplug the dividers of the breeding tanks, and allow the fish to breed for 10-15 min. Collect the eggs using a strainer, and examine the cell stage and quality of the eggs under a stereomicroscope. Select the eggs in the single-cell state for injection to improve the editing efficiency.
7. Line the eggs on a 1.5% agarose injection plate, and inject 2 nL of the mixture into the cell, not the yolk, of the embryos under a microscope to improve the editing efficiency. Retain at least 15 eggs as the control group.
8. Use 1x E3 buffer to collect the eggs in Petri dishes, and place them in an incubator at 28 °C. Remove the dead eggs, and change the culture buffer every 12 h until 48 hours post fertilization (hpf).

4. Efficiency analysis of base editing with EditR

1. At 48 hpf, collect three pools of six randomly selected injected embryos and six randomly selected control embryos into PCR tubes respectively. Use a pipette to aspirate the residual E3 buffer.
2. Add 40 µL of 50 mM NaOH into the PCR tubes. Put the tubes in a metal bath at 95 °C for 20 min, and then vortex for 15 s.
3. Add 4 µL of 1 M Tris·HCl (pH 8.0) into each tube to neutralize the NaOH. Briefly centrifuge at 2,000 x g for 10 s at room temperature to remove droplets from the tube wall. Collect the supernatant into new PCR tubes, and store them at −20 °C.
4. Design appropriate primers upstream and downstream of the gRNA target sites. Use 1 µL of the above supernatant as template for the PCR reactions of 50 µL volume. The primers used for the PCR in this study are listed in Supplementary Table 1.
5. Perform a DNA agarose gel electrophoresis to ensure correct and specific bands, followed by Sanger sequencing as previously described⁴⁰.
6. Analyze the Sanger sequencing data with the EditR (1.0.10) program⁴¹.
   1. Upload the sequencing file (ab1 format) into the "Upload .ab1 File" box.
   2. Enter the 20 bp gRNA sequence in 5′-3′ orientation into the "Enter gRNA sequence" box.
   3. Open the sequencing file (ab1 format), and confirm the base positions where the 20 bp gRNA sequence starts and ends. Enter the corresponding base position in the "5′ start" and "3′ end" boxes.
   4. Click on Predicted Editing to obtain the base editing efficiency. Check the quality of the sequencing data near the gRNA target sequence to avoid disorderly peaks or indels.
      NOTE: In general, the disorderly peaks can be effectively removed by adjusting the PCR annealing temperature.

Wyniki

The mutation of TSR2 has been reported to cause Diamond Blackfan anemia (DBA)⁴². Here, a DBA zebrafish model was constructed with a tsr2 (M1V) mutation using the i-Silence strategy. The adenine of the start codon of the zebrafish tsr2 was successfully converted to guanine using zSpRY-ABE8e (Figure 3).

The EditR analysis of the Sanger sequencing results showed that there was an A/G overlap at the adenine base of th...

Dyskusje

This protocol describes the construction of a zebrafish disease model using the base editor zSpRY-ABE8e. Compared with the traditional HDR pathway for base substitution, this protocol can achieve more efficient base editing and reduce the occurrence of indels. At the same time, this protocol involves implementing the recently proposed i-Silence gene-knockout strategy in zebrafish. Taken together, zSpRY-ABE8e will enhance the application of zebrafish models in disease research.

Off-target effec...

Materiały

Name	Company	Catalog Number	Comments
Agarose	Sigma-Aldrich	A9539	1.5% Agarose used to make an injection plate
Borosilicate Glass Capillaries	Harvard Apparatus	BS4 30-0016
Cell culture dishes	Falcon	351029
ClonExpress Ultra One Step Cloning Kit	Vazyme	C115	Kit for Infusion clone
Codon optimization service	Sangon Biotech
Drummond Microcaps	Drummond Microcaps	P1299-1PAK	Length:32 mm, capacity:0.5 μL
EasyEdit gRNA service	GenScript
Fine Forceps	Fine Scientific Instrument	11254-20	Used to break meedle
Flaming/Brown Micropipette Puller	Stutter Instrument	P-97	Used to pull the glass capillaries
HotStart Taq PCR StarMix	Genstar	A033-101	Used for PCR reaction
Intelligent artificial climate box	TENLIN	PRX-1000A	Used to culture zebrafish embryos
Methylcellulose	Sigma-Aldrich	M0512	Used to fix zebrafish when photographing
Microloader pipette tips	Eppendorf	5242956003
mMACHINE kit
Mut Express II Fast Mutagenesis Kit V2	Vazyme	C214-01	Kit for site-directed mutagenesis
Pneumatic Microinjector	ZGene Biotech	ZGPCP-1500 PLUS
pT3TS-zSpCas9	Addgene	46757
RNeasy FFPE kit	Qiagen	73504	Kit for RNA purification
Sanger Sequencing service	Sangon Biotech
Sodium hydroxide, granular	Sangon	A100173-0500	NaOH used for genome extraction
Stereo Microscope	Olympus	SZX10	Used for photograph of phenotype
SZ Series Zoom Stereo Microscope	CNOPTEC	SZ650
T3 mMESSAGE	Ambion	AM1348	Kit for in vitro transcription
TIANprep Mini Plasmid Kit	TIANGEN	DP103-03	Kit for plasmid extraction kit
TIANquick Mini Purification Kit	TIANGEN	DP203-02	Kit for purification for linearized plasmid
Tricaine	Sigma-Aldrich	E10521	Used to anesthetize zebrafish
Tris (hydroxymethyl) aminomethane	Sangon	A600194-0500	Component of Tris·HCl used for genome extraction
XbaI	New England Biolabs	R0145S	Restriction endonuclease used for plasmid linearization