Elsevier

Methods

Volume 69, Issue 1, 15 August 2014, Pages 67-75
Methods

TALEN construction via “Unit Assembly” method and targeted genome modifications in zebrafish

https://doi.org/10.1016/j.ymeth.2014.02.010Get rights and content

Abstract

Transcription activator-like effector nucleases (TALENs) are engineered endonucleases composed of a customized transcription activator-like effector (TALE) DNA-binding domain and a FokI DNA cleavage domain. TALENs induce DNA double-strand breaks (DSBs) at their target sites on the chromosome and have been successfully used for genome engineering in many species and cultured cells. Zebrafish is a very popular model organism in both basic and clinical research. Here, we describe the details of construction of customized TALENs using the “Unit Assembly” (UA) method, as well as three applications of zebrafish genome manipulations using TALENs: gene knock-out, large chromosome deletion, and gene knock-in by homologous recombination.

Introduction

Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly-interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system are emerging reverse genetic tools for targeted genome modifications, and all have been successfully applied to zebrafish (Danio rerio) [1], [2], [3], [4]. These technologies enable researchers to elucidate the functions of genes of interest in zebrafish development and generate disease models for clinical research. Although a high-throughput study has identified thousands of zebrafish mutants [5], it is difficult to achieve saturated mutation or precise genome modification using forward genetic approaches. With the publication of a high-quality genome of the zebrafish [6], it is now possible to study the functions of each gene using these reverse genetic approaches.

In our previous studies, we reported the “Unit Assembly” (UA) method to assemble transcription activator-like effector (TALE) repeats [7]. With this method, the TALEN technology is applied to targeted genome modification in zebrafish, achieving gene knock-out [7], large genomic deletions [8], [9], and gene knock-in through homologous recombination [10]. UA is based on alternative TALE repeats flanked by two isocaudamer restriction enzymes, NheI and SpeI. The advantages of UA are simple in strategy, ease of handling (all the steps are solely based on standard molecular cloning experiments), cheap for construction, and reliable in performance. In addition, all the intermediate products (plasmids and DNA fragments recovered from gel extraction) can be saved and reused in further constructions to save time and materials. Other methods can also be used to construct TALE repeats, such as Golden Gate derivate methods [11], [12], [13], [14], [15], [16], [17], REAL (restriction enzyme and ligation) [18], [19], FLASH (fast ligation-based automatable solid-phase high-throughput) [20], ICA (iterative capped assembly) [21], and LIC (ligation-independent cloning technique) [22]. UA and REAL are based on standard molecular cloning methods, which use a few starting materials (e.g., four single TALE repeat plasmid plus two FokI vectors) and TALEN expression vectors can be constructed in one to 2 weeks by several rounds of restriction enzyme digestion-ligation steps with high fidelity. Golden Gate derived methods need more starting materials, but these methods enable users to assemble multiple TALE repeats together in a single ligation reaction and can theoretically finish TALEN construction within 1 week. FLASH and ICA are solid-phase based modular assembly methods, which enable users to make large number of TALEN constructs simultaneously by using magnetic beads and multi-channel pipets or automated robots. Because of the development of these methods, TALE construction is no longer a bottleneck for the application of this technology.

TALENs cleave the chromosome at their target sites and create DNA double-strand breaks (DSBs) (Fig. 1A). When the DSB is repaired by the error-prone non-homologous end-joining (NHEJ) pathway, small indel (insertion/deletion) mutations can be introduced at the target sites. This can lead to frame-shifts and disrupt the functions of coding genes. In addition to gene knock-out, applying two pairs of TALENs can delete large chromosome regions between the two target sites [8]. Large chromosome deletion is useful to study the functions of non-coding genes, gene clusters or gene regulatory elements. It also can be used to simultaneously disrupt multiple adjacent genes or genes with multiple transcripts, splice variants, or alternative translation initiation site, which are difficult to be completely disrupted by the small indel mutations. To exclude the un-controllable and un-predictable side-effects of truncated proteins caused by indel mutations, the strategy of chromosome deletion can also be used as an alternative way to mutate a single target gene by deleting its coding region or important motif(s) or domain(s), or even the entire gene [9]. DSBs have been shown to be able to increase the efficiency of homologous recombination (HR) in the presence of a template [10]. Precise genome manipulation can be achieved through homologous recombination, such as gene knock-in, conditional knock-out, and making point mutations (Fig. 1B).

Here, we describe the details of how TALENs are constructed using UA. We also introduce the application of TALENs to edit the zebrafish genome by gene knock-out through NHEJ, large chromosome deletion using two pairs of TALENs, and gene knock-in through homologous recombination.

Section snippets

For zebrafish husbandry and breeding

  • (1)

    Wild type Tübingen fish or other desired zebrafish strains.

  • (2)

    Mating tanks (ESEN, China).

  • (3)

    E3 embryo buffer (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4).

  • (4)

    Stereo microscope (Zeiss, Germany).

  • (5)

    MPPI-2 Pressure Injector (Applied Scientific Instrumentation, USA), or PLI-90 Pico-Injector (Harvard Apparatus, USA).

  • (6)

    Glass capillaries for making injection needles (O.D. 1.0 mm, I.D. 0.58 mm; Harvard Apparatus, USA).

  • (7)

    PN-30 Puller (Narishige, Japan).

  • (8)

    28.5 °C incubator (Sanyo, Japan).

For the construction of TALEN vectors

  • (1)

    UA method starting vectors.

    4

Selection and validation of TALEN target site

Since TALENs function as dimers, a complete TALEN target site consists of a left TALEN binding site, a spacer, and a right TALEN binding site (Fig. 1A). TALEN target sites can be picked up manually or using web-based programs, such as TALE-NT (http://tale-nt.cac.cornell.edu/) [23], ZiFiT (http://zifit.partners.org/ZiFiT/) [24], and E-TALEN (http://www.e-talen.org/) [25]. For convenience in experiments (i.e., efficiency evaluation and mutant screening), target sites that have a unique

TALEN mRNA preparation by in vitro transcription

For in vitro transcription experiments, all the reagents and equipments must be RNase-free. The pCS2-TALEN plasmids are linearized by NotI (located downstream of the TALEN coding region) and used as templates to synthesize mRNA. To generate capped mRNA encoding TALENs, the SP6 mMESSAGE mMACHINE kit (Ambion) is used for in vitro transcription following the manufacturer’s instructions. The mRNA is purified using the RNeasy Mini kit (Qiagen). After quantification by NanoDrop spectrophotometer, the

Overview of chromosomal deletion

TALEN-induced indel mutations in early exons are suitable for knocking out some protein-coding genes. However, for other complex situations, the indel mutation strategy is not sufficient. For example, to disrupt a non-coding gene or a transcription cis-element, it is better to delete the whole region rather than induce a small indel in these non-coding sequences. The chromosomal deletion strategy is also applicable to the disruption of gene clusters, genes with multiple transcripts or splice

Overview of HR

HR can precisely modify the genome, which is essential to study the functions of genes and to develop models of human diseases in mice and zebrafish. Spontaneous HR occurs at a very low frequency, and it is not practical in zebrafish where embryonic stem cells are still not available. The site-specific DSBs have been reported to be able to stimulate HR dramatically in many organisms. Thus, TALEN technology could be used to introduce DSBs to achieve HR in zebrafish [10], [32].

Selection of TALEN target sites for HR in the target gene

The selection of

Acknowledgements

We thank Ian Bruce for language editing of our manuscript; Yan Shen and Yuying Gao for the management of TALEN constructs; Yingdi Jia and Jingliang Chen for zebrafish maintenance; Da Liu for help to illustrate figures; Zhenchao Cheng and Yingdan Wu for help to edit the manuscript; and all the members in our lab for their efforts on optimizing the TALEN applications. This work was partially supported by the 973 Program of the Ministry of Science and Technology of China (2012CB945101, 2011CBA01000

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    1

    Present address: Division of Hematology, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA.

    2

    Present address: Department of Internal Medicine, Eastern Virginia Medical School, Norfolk, VA 23507, USA.

    3

    These authors contributed equally to this work.

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