Elsevier

Methods

Volume 69, Issue 1, 15 August 2014, Pages 32-37
Methods

A simple TALEN-based protocol for efficient genome-editing in Drosophila

https://doi.org/10.1016/j.ymeth.2014.03.020Get rights and content

Abstract

Drosophila is a well-established genetic model organism: thousands of point mutations, deficiencies or transposon insertions are available from stock centres. However, to date, it is still difficult to modify a specific gene locus in a defined manner. A potential solution is the application of transcription activator-like effector nucleases (TALENs), which have been used successfully to mutate genes in various model organisms. TALENs are constructed by fusion of TALE proteins to the endonuclease FokI, resulting in artificial, sequence-specific endonucleases. They induce double strand breaks, which are either repaired by error-prone non-homologous end joining (NHEJ) or homology directed repair (HDR). We developed a simple TALEN-based protocol to mutate any gene of interest in Drosophila within approximately 2 months. We inject mRNA coding for two TALEN pairs targeting the same gene into embryos, employ T7 endonuclease I screening of pooled F1 flies to identify mutations and generate a stable mutant stock in the F3 generation. We illustrate the efficacy of our strategy by mutating CG11617, a previously uncharacterized putative transcription factor with an unknown function in Drosophila. This demonstrates that TALENs are a reliable and efficient strategy to mutate any gene of interest in Drosophila.

Introduction

Drosophila melanogaster is a classical model to investigate gene function in a complex, multi-cellular organism. Starting with the legendary Heidelberg screen for embryonic patterning [1], [2] and followed by hundreds of its derivatives, forward genetic screens have been immensely powerful to discover genes and study their functions in Drosophila biology (reviewed in [3]). Forward screens per definition hit the genome at random and thus discover new roles for genes in a particular biological context.

Reverse genetics, on the other hand, tests gene function by a targeted approach manipulating one particular gene of interest. After 30 years of forward screens, reverse genetics is currently booming in Drosophila, as many large scale systematic approaches such as interaction proteomics [4] or genome-wide RNAi screens [5], [6], [7] suggest a function for a given gene without providing classical loss of function mutations. Tools to apply reverse genetics in Drosophila have advanced rapidly in the last years, in particular through gene targeting by homologous recombination that allows gene modification at the endogenous gene locus, similar to the approach in mice [8], [9]. However, despite significant progress in the gene targeting protocol enabling smart genetic selection methods [10], it remains relatively tedious to engineer a particular gene of interest to either a genetic null allele or a tagged protein allele. The recent implementation of zinc-finger nucleases in Drosophila opened a promising new path for gene engineering [11], [12], but zinc-finger design is laborious and has certain sequence restrictions. Mixed-success reports, most likely dependent on the target sequence [13], have prevented the technology from taking off as a routine application for generating mutants in the average fly lab.

Transcription activator-like effector nucleases (TALENs) are derived from naturally occurring transcription factors present in plant pathogens called TALEs, which contain 12–27 repeats, typically composed of 34 amino acids. Each repeat binds to a single base pair of DNA, with the two central variable amino acids determining base specificity [14], [15]. In TALENs, the C-terminal transcriptional activation domain has been replaced by the FokI nuclease domain, converting a transcriptional activator to a sequence specific nuclease. As FokI cleaves only as a dimer, TALENs are used in pairs that bind on opposite DNA strands, separated by a central spacer domain. TALEN-induced double strand DNA breaks are either repaired by non-homologous end joining (NHEJ), which often results in small insertions or deletions and is thus valuable to mutate genes, or by homology directed repair (HDR) using the homologous chromosome or a provided homologous DNA sequence, which can be used for gene engineering [16]. This strategy has been applied to a number of different organisms or cell types, including Arabidopsis, zebrafish, and human cell lines, to mutate genes or insert DNA sequences into a locus of interest [17], [18], [19], [20]. In addition to their straight forward application, TALENs are more flexible than zinc-fingers as they can recognise any DNA sequence and also appear to have higher target specificity and lower toxicity [19], [21], [22].

In Drosophila, TALENs have mainly been used to mutate genes that result in visible phenotypes such as changes in eye or body colour. Thus, they can easily be scored in the F1 generation [22], [23], [24]. Lately, this technology has been expanded to also mutate genes with unknown phenotypes in Drosophila [22] or to insert GFP into desired locations in the fly genome [24], demonstrating the potential of TALEN-mediated genome editing. Here, we report a simple and effective protocol to apply TALEN-mediated genome engineering in order to mutate any gene of choice in Drosophila. Our protocol can easily be performed by a standard Drosophila laboratory without the need to purchase special equipment. We generate mosaic G0 adult flies by injecting Drosophila embryos with mRNAs coding for two pairs of TALENs that target next to each other in the same gene region. We apply a T7 endonuclease I screening assay to detect heterozygous mutant carriers amongst the F1 generation and generate a mutant stock using the F3 generation. Together, this allows the effective generation of mutations in any Drosophila gene within about 2 months.

Section snippets

Cloning of TALEN pairs

  • Addgene TALEN kit (Addgene).

  • pCS2TAL3-DD/RR backbone plasmid (Addgene).

  • QIAprep Spin Miniprep Kit (Qiagen).

  • QIAGEN Plasmid Midi Kit (Qiagen).

  • Bsa I and BsmB I (NEB).

  • T4 ligase (NEB).

  • Plasmidsafe nuclease (Epicentre).

  • Tetracycline 10 μg/ml (Sigma–Aldrich).

  • Spectinomycin 50 μg/ml (Sigma–Aldrich).

  • Ampicillin 100 μg/ml (Sigma–Aldrich).

Plasmid linearisation and in vitro transcription of TALEN pairs

  • Not I (NEB).

  • 10% SDS (Sigma–Aldrich).

  • Protease K (Sigma–Aldrich).

  • Phenol/Chloroform/Isopropanol (v:v:v = 25:24:1).

  • 3 M NaOAc (pH 5.2).

  • RNase free Eppendorf tubes (1.5 ml) and tips.

Application of TALENs to Drosophila

Fig. 1 illustrates the different steps in our protocol, starting with design of the TALEN target sequences and ending with the final mutant Drosophila stock. The entire protocol can be completed in about 2 months.

Results and discussion

We applied our protocol to generate null alleles of the putative Zn-finger transcription factor CG11617 (Fig. 3-1). CG11617 is located at 21B4 on the second chromosome and mutant alleles have not been described. Following the above protocol, we designed two TALEN pairs targeting the first coding exon of CG11617 (Fig. 3-2). We injected a mixture of all four TALEN mRNAs into 400 white1118 embryos, resulting in 36 fertile G0 flies that we crossed with If/CyO males or virgins. Using the T7

Acknowledgements

We are very grateful to Bettina Stender and Nicole Plewka for excellent technical assistance. We thank Valeria Soberon for initial help with the Golden Gate system and Maria Spletter for critical comments on the manuscript. This work was supported by the Max Planck Society, the European Research Council and a Career Development Award by the Human Frontier Science Programme to F.S.

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