Drosophila CRISPRi/CRISPRa Service
Cas9 is a powerful DNA-binding unwinding protein that may assists other proteins turn transcription on or off dynamically and quantitatively to regulate targeted gene expression. CRISPRi/CRISPRa is a more flexible tool, especially in vivo that effectively prevents the cytotoxicity caused by double-stranded DNA breaks, and compensates for the limitations of CRISPR/Cas9 permanent knockout of essential genes and long non-coding RNAs, etc. Therefore, CD BioSciences offers genome-wide CRISPR strategy in the fruit fly field, which is also a powerful complement to RNAi and overexpression limited to a few genes or tissues. A non-catalytic Cas9 (dCas9) fusion protein guided by sgRNA is used to target the effector domain to specific DNA sequences to inhibit (CRISPRi) or activate (CRISPRa) transcription genes.
CD BioSciences as an experienced Drosophila scientists project management team, focuses on the critical decisions needed for cutting-edge biological science research. We are a trusted, indispensable supporter to our customers, providing cost-effective and complex holistic services. Our CRISPRi and CRISPRa services conduct clients robust whole-genome modulation of transcription by designing dCas9 protein and unique sgRNA of endogenous genes.
|CRISPRi||dCas9-sgRNA complex targets the promoter or coding sequence of the target gene, temporarily blocking RNA polymerase and gene transcription.||Construction of CRISPRi components, conditional expression dCas9 (or dCas9-VPR) Drosophila strains, sgRNA plasmids or strains at whole-genomic level, efficiency assessment, etc.|
|CRISPRa||Gain-of-function (GOF) transfer strategy. dCas9 fusing transcriptional activators (VPR, SAM, etc.), binds to the target gene transcription start site (TSS) with sgRNA, activating gene overexpression.|
Fig.1. Drosophila CRISPRi/CRISPRa
Workflow of Drosophila CRISPRi/CRISPRa
- CRISPRi/CRISPRa Strategies
- sgRNA and dCas9 Constructs
- Phenotypic Detection and Validation
- Evaluation of the Efficiency of CRISPRi/CRISPRa System
From our professional platform, robust and complete strategies are designed, involving target sites, sgRNA sequences, dCas9 or dCas9 variants, etc. The success rate of a single sgRNA against CRISPRi/a varies greatly, so we generally design dual-sgRNAs per target gene to maximize the success rate. Bioinformatic prediction of off-target effects ensures high specificity and efficiency of sgRNAs.
We generate efficient the vector or proteins components needed for on-target editing. Both sgRNA and dCas9 co-expression plasmids and transgenic Drosophila strains are available. sgRNA and dCas9 expression plasmids for Drosophila in vivo may be confirmed first in vitro.
In generally, an exogenous marker or reporter helps identify candidates. But there are no limits in screening. We look at the success rate of regulation in each transgenesis by qPCR or further biochemical analysis (optional). And for any transgenic issue, sequence validation is necessary in our evaluation system.
We first check whether the CRISPR strategy resulted in mass deaths of genetically modified fruit flies. If the survival rate is significantly reduced, we will redesign the process.
We promise to offer highly specific genome-scale CRISPRi/ CRISPRa service with low off-target activity and complete experimental parameters and analysis.
We provide regular updates and in-time messages if any issue or complication happen during the manufacturing process.
What CD BioSciences offers
Professional and experienced team accelerates clients' research timeline with a range of standardized and tailored services.
- We provide a comprehensive and extensive CRISPRi/a system characterization platform with common and "visible" strains stock to evaluate the usability of each CRISPRi/a combination.
- Our design service contains not only co-expression vectors but also sgRNA and dCas9 transgenic strains. We will try our best to meet customers' needs.
- We provide dual sgRNA library construction with high-specificity and high-efficiency.
- Tailored complex CRISPRi/CRISPRa strategies are served at the same time, for example, high-throughput screening, spatio-temporal regulation, clonal reporter system, etc.
- One-stop further services for exploring and validating gene function involving our other Genetic, Cell Biology and Biochemistry are available on our website.
- Long non-coding RNAs function
- The function of related metabolic network
- High-throughput whole-genome screening
- Study the role of transcriptional regulatory elements
CD BioSciences as a global biotechnology research-driven compony, implementing a well-effective Drosophila transgenic strategy is imperative to expedite your trials timelines, keep track of project milestones and navigate Genetic and Cell Biology research. Our services are not more than just CRISPR/Cas9 transgenes. CRISPRi/CRISPRa genome-scale libraries construction and spatio-temporal CRISPRi/CRISPRa services are also provided. Contact us to learn more about our related services to start your fly's exploration.
- Gilbert LA, et al. (2014). Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell. 159(3), 647–661.
- Ewen-Campen B, et al. (2017). Optimized strategy for in vivo Cas9-activation in Drosophila. PNAS. 114 (35) 9409-9414.
For research use only. Not intended for any clinical use.