Optimized TALEN Application and Screening in Drosophila melanogaster

Justin Overcash, Graduate Research Assistant, Entomology, Virginia Tech,
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Lee et al 2015 provide a detailed methodology for gene editing using TALENs in Drosophila melanogaster. While TALEN use is relatively well established in D. melanogaster, fine tuning the injection concentrations of designer nucleases allows for the maximum efficiency in regards to survival, fertility, and genetic modification rates.  While CRISPR/Cas9 is becoming increasingly popular as a genome editing tool, TALENs should not be discounted or underrated.  So Lee et al’s paper should be of general interest.

Using TALEN mRNA concentrations between .5 µg/µl and 4.3 µg/µl they show that TALEN mRNA levels are directly related to survival, fertility and gene editing rates. While injection mixes containing lower concentrations (.5 µg/µl) of TALEN mRNA have higher survival and fertility rates, no G0 somatic mutations were observed. Conversely, higher concentrations lead to readily observable somatic mutations but lower survival and fertility rates.

Structure of a TALE binding to DNA

Structure of a TALE binding to DNA

Lee et al was also able to establish that mutation rates observed in G1 progeny is TALEN-dosage dependent. This suggests that while higher survival and fertility rates are optimal, the negative impact of utilizing higher mRNA concentration may be counteracted by the appearance of more germline mutations.

Lastly, Lee et al has developed a unique assay used to measure bi-allelic mutations in Drosophila (even in the absence of an easily observable phenotype). This assay allows the experimenter to quickly assess the efficiency of TALEN pairs and CRISPR sgRNAs. While assays such as high resolution melt curve analysis (HRMA) and restriction fragment length polymorphism (RFLP) are currently used for the detection of gene editing efficiency, the purposed assay does not rely on PCR and therefore provides a quick and cheap alternative assessment of designer nuclease efficiency.

Ligase IV plays a central role in Classical Non-Homologous End-Joining.

Their assay is survival based and utilizes a Lig4 knockout line. Lig4 plays a crucial role in the ligation complex within the classical non-homologous end joining DNA repair pathway (C-NHEJ). Its removal significantly impacts the ability for a cell to repair via C-NHEJ and has been shown to shift the DNA DSB repair hierarchy (Beumer et al 2013). They hypothesized that with a reduction in C-NHEJ in combination with a lack of template for homology directed repair (due to bi-allelic mutations) a higher rate of embryonic death would be directly linked to increased nuclease efficiency.

Using both a known efficient TALEN pair and an inefficient TALEN pair, Lee et al was able to show that the Lig4 null line can indeed be utilized to measure TALEN efficiency. Similarly, Lee et al tested the CRISPR/Cas9 system and gained similar results, suggesting that TALEN and sgRNA efficiency can be gauged with Lig4 mutant lines.

DmelLee et al has provided an in-depth assessment of TALEN mRNA concentration on survival, fertility and gene editing rate. They also have devised a clever way to quickly measure the efficiency of designer nucleases. While this assay is dependent upon the ability to establish a Lig4 null line, if a Lig4 null line is possible in your organism of choice, it may provide a cheap and easy way to screen potential designer nucleases.

 

Lee HB, Sebo ZL, Peng Y, Guo Y. An optimized TALEN application for mutagenesis and screening in Drosophila melanogaster. Cellular logistics 2015; 5(1): e1023423.

Beumer KJ, Trautman JK, Mukherjee K, Carroll D. Donor DNA Utilization during Gene Targeting with Zinc-finger Nucleases. G3 2013.

 

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