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01 / Non-Drosophila melanogaster Gene Editing
Fungene Bio has successfully obtained CRISPR gene-edited strains in multiple non-Drosophila melanogaster species, including D. simulans, D. yakuba, D. virilis, and D. suzukii.
Technical Process

① Scheme Design
② Sample Preparation
③ Microinjection
④ Crossbreeding and Identification
⑤ Construction of Homozygous Lines

Technical challenges and solutions compared to Drosophila melanogaster

Technical Challenges

Solutions

Breeding conditions differ from D. melanogaster; poor growth under standard conditions.

Developed various media for optimal breeding of different fruit fly species.

Uncertain gene editing efficiency

Efficient gRNA/cas9mRNA to enhance gene editing success rates.

Lack of balancer chromosomes makes it difficult to establish homozygous lines from positive flies.

Established mature and efficient identification and hybridization methods for rapid acquisition of homozygous lines.

Instability in preserving mutant genes if homozygous lines cannot be obtained

Molecular identification while ensuring fly survival, effectively enriching heterozygous mutant lines.


Service Process
The specific service process and cycle are as follows, inquiries are welcome.
Experimental StepsContentDuration (months)
Target SequencingBy sequencing, make sure that there are no mutations on the targets0.5
Synthesis of gRNA and cas9mRNAIn vitro transcription and purification of 2gRNAs(for one target)
In vitro transcription and purification of cas9-mRNA
Make the injection mix(gRNAs+cas9-mRNA)
MicroinjectionThe injection mixture consists of gRNA, cas9-mRNA, and donor, and 500-600 embryos will be injected.Variable, based on the life cycle, 1-2 generations
Cross、Molecular IdentificationP0 activity test for positivity, cross with WT, confirm positive rate by PCR sequencing.Variable, based on the life cycle, 3-4 generations + identification time
Positive Identification and HybridizationScreen positive P0 F1 offspring, interbreed positive adults; if few F1 positives, cross with WT to amplify, then interbreed F2 adults.
Construction of Homozygous LineIdentify homozygotes in F2 (or F3), interbreed positive homozygotes; if not homozygous, deliver heterozygous lines.
Total
02/ Gene Editing for Other Insects
Fungene Biotech is committed to building and developing gene editing platforms for other insect species. We aim to leverage our experience and advantages in gene editing technology, in conjunction with the client's breeding experience, to continuously develop and establish new gene editing platforms for various insect species.
Technical Process

① Feasibility Assessment: Research literature to gauge editing complexity and assess project feasibility.
② Breeding and Injection Pilot Experiments: Optimize breeding and injection to achieve reliable results.
③ Official Project Initiation and Execution: Apply preliminary methods and processes to initiate the project and obtain the edited strains.

Success Stories





                 B. dorsalis                      M. domestica                      L. migratoria                      O. furnacalis