Drosophila TALEN Genome Editing
The most modern tool for modifying complex genomes at the single nucleotide level is site-specific nucleases, which can induce DNA double-strand breaks (DSBs) at specific genomic locations. The repair pathway triggered by DSB can trigger targeted gene reprogramming, mainly through two mechanisms: error-prone non-homologous end joining (NHEJ) and precise homologous directed recombination or repair (HDR).
Transcription activator-like effector nucleases (TALEN) is one of the efficiency tools in genomic editing. Compared with ZFN, TALENs are much easier to design and build. Due to high target specificity, there is low off-target DNA cleavage. Moreover, the cytotoxicity is reduced to a certain extent. TALEN and CRISPR/Cas9 systems are quickly adopted by biologists due to their high efficiency and easy-to-operate procedures in DSB induction, including targeted gene modification in basic Drosophila theoretical research and applied research (such as cancer and drug research).
CD BioSciences is a global research company focused on Drosophila science solutions. Relying on the mature fruit fly transformation technology platform, we provide customized TALEN genome engineering service for customers, and solve the technical challenges of TALEN. As your supporter, we will escort your research projects and let you focus on the essence of scientific research.
- Feature Service
TALEN technology combines various homologous donor DNA plasmids to manipulate the Drosophila genome: (1) precise generation of genomic deletions; (2) DNA fragment replacement at the single nucleotide level; (3) precise tagging to track endogenous protein expression.
TALE protein (Transcription activator-like effector) was first detected in the plant pathogen Xanthomonas spp. It was found to activate the transcription of specific genes in plant cells during infection by pathogens. TALE protein usually consists of 3 domains: N-terminal nuclear localization signal domain, C-terminal domain with transcriptional activation function, and tandem repeat domain that can recognize specific DNA sequences. The tandem repeat domain contains a wide range of repeating elements, from 5 to more than 30, with an average of 17.5. Each TALE repeat is composed of 34 amino acid residues, of which the 12th and 13th are highly variable called repeat-variable diresidues (RVDs), corresponding specific nucleotides (NI-A, NG-T, NN/NK-G, HD-C). The C-terminus of the TALE protein combining with the Fok I domain forms TALEN. A pair of TALENs binds to the target DNA sequence, thus generates DSB and obtains gene mutants.
Fig.1 Mechanism of TALEN genome editing (Li HY et al. 2020)
- TALEN Strategy
The key of TALEN genomic editing technology is constructing a highly specific TALEN assembly and an appropriate TALEN injection method to reduce the workload of screening mutant offspring. Our experienced and professional technical team customize efficient and specific TALEN components for your scientific research needs, greatly reducing your time to generate fly collections, allowing you to focus on your scientific research projects. At the same time, we provide you with a one-stop TALEN gene editing related analysis service, and issue a detailed result report, including experimental design and original data.
- Construction of TALEN Vector and Donor DNA
We provide TALEN DNA plasmid or TALEN mRNA assembly solutions. As for TALEN construction, we recommend the length of TALEN-binding sequence to be between 12 and 17 bp, based on the balance of binding specificity and practical construction of the TALEN pairs using Unit Assembly. The reported spacer sequence for functional TALEN pairs normally ranges from 13 to 30 bp. For different purposes and efficiency, this number will be adjusted accordingly. If you choose to inject TALEN mRNA directly, we have an additional mRNA in vitro transcription and modification step. In addition, we have complete platform of assembling the DNA binding arrays within a short time.
The use of HDR-mediated gene editing requires homologous DNA. The donor DNA with homology arms is designed for the use of nucleotide substitutions, deletions, tag insertions, etc., and is cloned into the donor plasmid. The typical homology arm length is 1.5 kb, but different experiments have different lengths.
Both TALEN assembly and donor DNA plasmid will be purified to be in good-quality, especially to be completely free of RNase.
- Screening and Isolation
Once fly embryo injection is completed, larvae will be raised to adulthood, crossing with balancer flies. And collecting offspring crossed with balancer flies to acquire stable mutants. In generally, an exogenous marker or reporter helps identify candidates. But there are no limits in screening. As standard, genetic screening by PCR and sequencing for each transgenesis.
The final Drosophila lines will be shipped with validation report, which describes the details of TALEN genome editing and shows evidence of the required editing.
- TALEN Efficiency Evaluation (Optional)
We provide a variety of methods to evaluate the efficiency of custom-made nuclease-induced site-specific mutagenesis: restriction fragment length polymorphism (RFLP), high-resolution melting analysis (HRMA), single-strand annealing (SSA) assay, etc.
There are several factors that affect the efficiency of TALEN pairs, including TALEN mRNA concentration, chromatin accessibility at the locus, polymorphism of TALEN DNA binding site, and external experimental conditions.
We are a team of professional doctoral scientists led by technology, and have many years of experience in the field of Drosophila TALEN genetic transformation. A series of strict quality control platforms have been established to customize high-quality and transparent scientific services for clients.
- We offer specially-customized TALEN genome editing services, including loss of function mutations (frameshift or large deletion mutations), insertion of foreign sequences, such as recombinase integration sites and reporters, as well as single-nucleotide level correction for gene therapy theory.
- In view of the different principles of DSB repair, we design different donor plasmids and TALEN assemblies for HDR and NHEJ-mediated TALENs, and customize the most simple and effective screening strategy.
- Although TALEN cannot be used to construct large-scale mutant libraries, the TALE repeat targets a single genomic site. Fusions of TALE and other effector domains can be designed, such as transcription activators and site-specific recombinases, for the purpose of regulating protein expression and other genomic manipulations.
Drosophila in vivo TALEN genome editing platform has a broad usage in fundamental research and disease mechanism.
- Protein research.
- Metabolism research.
- Development and cell biology research.
- Drug targets research.
- Insect management research.
CD BioSciences relying on advanced TALEN technique operates fruit fly's whole-genome editing with less off-target effect. With a transparent price and an experienced team, we devote into offering a global, fast turnaround and high-quality Drosophila TALEN platform, as well as analysis services. If you have any questions or want to make a reservation service, you can contact us directly from our website, and we will respond in a short time.
- Lee HB, et al. (2015). An optimized TALEN application for mutagenesis and screening in Drosophila melanogaster. Cell logist. 5(1), e1023423.
- Liu J, et al. (2016). Methods for TALEN-mediated genomic manipulations in Drosophila. Methods Mol Biol. 1338, 179–190.
For research use only. Not intended for any clinical use.