Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis

Summary

This paper presents the step-by-step protocols for CRISPR/Cas9 mutagenesis of the Oriental fruit fly Bactrocera dorsalis. Detailed steps provided by this standardized protocol will serve as a useful guide for generating mutant flies for functional gene studies in B. dorsalis.

Abstract

The Oriental fruit fly, Bactrocera dorsalis, is a highly invasive and adaptive pest species that causes damage to citrus and over 150 other fruit crops worldwide. Since adult fruit flies have great flight capacity and females lay their eggs under the skins of fruit, insecticides requiring direct contact with the pest usually perform poorly in the field. With the development of molecular biological tools and high-throughput sequencing technology, many scientists are attempting to develop environmentally friendly pest management strategies. These include RNAi or gene editing-based pesticides that downregulate or silence genes (molecular targets), such as olfactory genes involved in searching behavior, in various insect pests. To adapt these strategies for Oriental fruit fly control, effective methods for functional gene research are needed. Genes with critical functions in the survival and reproduction of B. dorsalis serve as good molecular targets for gene knockdown and/or silencing. The CRISPR/Cas9 system is a reliable technique used for gene editing, especially in insects. This paper presents a systematic method for CRISPR/Cas9 mutagenesis of B. dorsalis, including the design and synthesis of guide RNAs, collecting embryos, embryo injection, insect rearing, and mutant screening. These protocols will serve as a useful guide for generating mutant flies for researchers interested in functional gene studies in B. dorsalis.

Introduction

The Oriental fruit fly, Bactrocera dorsalis, is a cosmopolitan insect pest species that causes damage to over 150 species of fruit crops, including guava, mango, Eugenia spp., Surinam cherry, citrus, loquat, and papaya¹. The damage caused in Guangdong Province (China) alone is estimated at over 200 million yuans. Adult females insert their eggs beneath the skin of ripening or ripened fruits, causing decay and abscission of the fruit, which decreases fruit quality and overall yield of the crop². Since adult fruit flies have great flight capacity and their larvae bore into the fruit skin, insecticides requiring di....

Protocol

1. Target design and in vitro synthesis of sgRNA

1. Predict the structure of target genes of interest and determine the boundaries between exons and introns via bioinformatic analysis of the B. dorsalis genome (software applications used here are listed in the Table of Materials).
   NOTE: BLAT²⁰ was used to search potential gene loci in the genome. The high-quality RNA-seq reads (transcriptome) were aligned to the acquired gene loci using Hisat2²¹. Samtools²² was used to generate the sorted bam files. The sorted bam files ....

Results

This protocol presents detailed steps for the development of B. dorsalis mutants using CRISPR/Cas9 technology, including representative results from gDNA selection, collecting embryos and microinjection, insect maintenance, and mutant screening.

The example of the target site of the selected gene is located in the third exon (Figure 1C). This site is highly conserved, and a single band was detected by gel electrophoresis for the DNA template for synthetic.......

Discussion

The CRISPR/Cas9 system is the most widely used gene editing tool and has various applications, such as gene threpy³⁰, crop breeding³¹, and basic studies of gene fuctions³². This system has already been applied for gene editing in various insect species and has served as an effective tool for functional gene studies in pests. The protocols we present here standardize the procedure of design and synthesis of guide RNAs, collecting embryos, embryo injec.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
6x DNA Loading Buffer	TransGen Biotech	GH101-01
Artificial climate chamber	ShangHai BluePard	MGC-350P
AxyPrep Genomic DNA Mini-Extraction Kit	Axygen	AP-MN-MS-GDNA-250G
BLAT	NA	NA	For searching potential gene loci in the genome
Capillary Glass	WPI	1B100F-4
Eppendorf InjectMan 4 micromanipulator	Eppendorf	InjectMan 4
GeneArt Precision gRNA Synthesis Kit	Thermo Fisher Scientific	A29377
Hisat2	NA	NA	For aligning the transcriptome to the acquired gene loci
IGV	NA	NA	For visualizing the results from Transdecoder
Microgrinder	NARISHIGE	EG-401
Olympus Microscope	Olympus Corporation	SZ2-ILST
pEASY-Blunt Cloning Kit	TransGen Biotech	CB101-02	https://www.transgenbiotech.com/data/upload/pdf/CB101_2022-07-14.pdf
Phenol red solution	Sigma-Aldrich	P0290-100ML
Pipette cookbook 2018 P-97 & P-1000 Micropipette Pullers	Instrument Company		https://www.sutter.com/PDFs/cookbook.pdf
PrimeSTAR HS (Premix)	Takara Biomedical Technology	R040A
SAMtools	NA	NA	For generating the sorted bam files
sgRNAcas9-AI	NA	NA	sgRNA design http://123.57.239.141:8080/home
Sutter Micropipette Puller Sutter	Instrument Company	P-97
Trans2K DNA Marker	TransGen Biotech	BM101-02
Transdecoder	NA	NA	For combining the results of assemble transcripts and gene loci information https://github.com/TransDecoder/TransDecoder/releases/tag/TransDecoder-v5.5.0
TrueCut Cas9 Protein v2	Thermo Fisher Scientific	A36498
Ultra-trace biological detector	Thermo Fisher Scientific	Nanodrop 2000C