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

Summary

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

Abstract

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.

Introduction

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 regul....

Protocol

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 ....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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