Drosophila Conventional KO Service
CRISPR/Cas9-mediated gene knockout is directed by sgRNA to direct Cas9 protein to cut the target sequence of the target gene, resulting in double-strand breaks of the gene and destruction of the gene genome sequence. At present, the widely used Cas9 protein is derived from Streptococcus thermophilus, which plays an efficient role in Drosophila after being optimized by codons. This system shows accuracy, efficiency, and cost-effectiveness in a wider range of genetic modifications and editing.
CD BioSciences is a professional biotechnology company specializing in genomic engineering and analysis in model organism – Drosophila, contributing in Genomics and Functional Genomics. Equipped comprehensive sgRNA and Cas9 pool and star-of-the-art platform, we can knock out the targeted gene you interest in Drosophila by CRISPR/Cas9 technology and complete in 2-4 months. We guarantee delivery of at least 3 independent-transformed G0 or 3 F1 (either back-crossed to balancer or not) for the knockout. Additionally, we maintain your private vials in the facility as backup service.
Conventional CRISPR Knock-out Technology
A part of CRISPR system, sgRNA contains: 20bp target sequence without PAM structure (5 '-NGG-3') and loop skeleton of sgRNA is constructed. At present, the main ways to obtain sgRNA are as follows: (1) to obtain sgRNA by in vitro transcription driven by the prokaryotic promoter T7; (2) Pol III promoters U6 and H1 drive the expression of sgRNA in individuals or cells by direct injection or transfection of plasmid DNA. (3) Pol III or Pol II promoters drive tRNA-sgRNA expression, and tRNA is excised by RNA enzymes in vivo to release sgRNA.
Cas9 protein can also be obtained by in vitro transcription of the T7 promoter or by in vivo translation of plasmid DNA through an expression domain driven by Pol II promoters. Or we have a stock of Cas9-transgenic Drosophila for cross. Microinjection of plasmid expressing Cas9 protein and sgRNA can lead to deletion of genomic fragments or multi-site deletion, and improve individual survival and thus increase genetic conversion. Once the sgRNA and Cas9 protein both express in individual or cells successfully, sgRNA matches the specific DNA region, and RNA-guided endonuclease Cas9 cuts dsDNA targets complementary to the sgRNAs to carry out target gene knock out process.
Fig.1 Mechanism of Conventional CRISPR Knock-out (KO) Workflow of Conventional CRISPR KO
We have comprehensive CRISPR/Cas9 editing platform. Either stable genetic strains expressing Cas9 protein and sgRNA respectively or direct injection of Cas9 and sgRNA expression stains can be obtained in our service.
- CRISPR GENE KNOCK OUT DESIGN
- CRISPR COMPONENTS CONSTRUCTION
- DROSOPHILA INJECTIONS
- CANDIDATES SCREENING
- STRAIN CONFIRMATION
We collaborate with you to understand your conventional CRISPR KO expectations. Through our scientific platform, several optimal systems to maximize the chance of successful gene knock -out are designed for clients' choices. We follow your research process and adjust the strategy accordingly.
We deploy a variety of gene assembly approaches to get the exact and precise deletions. We have the ability to efficiently generate sequence and plasmids components required for injection mix components.
Our injection operators have professional skills. They have the ability of obtaining precision delivery of transgenesis reagents to Drosophila embryos. We perform a sufficient number of injections that promising >100 crossed G0. And raise the survived larvae to adulthood.
Often our process has a marker or reporter that helps us identify candidates, but there are no limits. For our genomic integrant, we look at each transgenesis by PCR and sequence.
Once we confirm the deletions have proceed as planned, cross the G0. F1 generation are positive screened and confirmed by full sequencing of the entire transgene sequence due to client's opinion. Balancing is necessary if the mutation is homozygous lethal or sterile. The certain transformants can be mailed to you either as adult flies or larvae. The final transformants vials are maintained in the facility for two weeks as backup.
As standard, we validate at the genomic level with advanced sequencing and we provide a functional readout that verifies gene KO efficiency.
During the Drosophila engineering phase, our team performs the desired edit(s) defined in the statement of work. Throughout the process, our project management provides on-time updates and will contact you immediately if any issue or complication arise during the process of implementation.
Application
- Study the biological functions of certain genes/proteins
- Function of non-coding clustered gene families
- Precise analysis of the role of different functional domains
- Build Drosophila mutant library
- Study the important signaling pathways in Drosophila
- Verify targets drug through Drosophila disease model
Why CD BioSciences?
- Record-proven expertise and experience both in genetic engineering and Drosophila.
- Consistent results and superior efficiency for high efficiency gRNA and specific Cas9, achieving ~100% on-target.
- Fast turnaround services to obtain your transformants in less time and lower price.
- Best tracking service with status updates outlining the on-time progress.
Aided by well-established, CD BioSciences has successfully completed numbers of gene deletion projects using CRISPR/Cas9. We provide you with phased progress and final report during the project implementation. The final report details every manufacturing process, including genetic transformation strategy, target vector design, construction and verification, genotyping assessment and sequence analysis. We will customize our offering to meet your specific project needs and protect your scientific investment. We are looking forward to serving you wholeheartedly and becoming your indispensable research support partner.
Because each project is different, if you don't see the CRISPR/Cas9 service you need above, please contact us for more information.
For research use only. Not intended for any clinical use.
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