A fast and reliable strategy to generate TALEN-mediated gene knockouts in the diatom Phaeodactylum tricornutum
Introduction
Diatoms are unicellular microalgae belonging to the Stramenopiles. They play an important role in global carbon fixation as well as for the nitrogen, phosphorous and silica cycles [1]. They are widespread in most aquatic habitats, where they need to cope with large variations of light quality and quantity [2], [3]. As diatoms may contain larger amounts of lipids (up to 50% of dry weight), which even can be increased by genetic manipulation [4], [5], they are suitable for the production of biodiesel and/or bioplastics [6], [7], [8]. The pennate Phaeodactylum tricornutum has become a model system for diatoms because of the availability of the genome sequence as well as genetic transformation techniques [9], [10], [11], [12], [13], [14], allowing reverse genetics approaches. Because of their diplontic life cycle and the lack of of sexual reproduction in the lab, many methods for genetic manipulation like random mutagenesis or crossing are not available. Accordingly, knockdown via RNAi is currently the most commonly used approach for genetic manipulations [15]. Recently, two new genetic tools for directed genome editing were developed, which allow induction of targeted DNA double-strand breaks to knock out genes irreversibly: the TALEN (Transcription activator-like effector nucleases) [16], [17], [18] and the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) systems [19], [20], [21]. Both approaches depend on nucleases that are guided to a specific DNA target sequence, and subsequently induce the formation of a DNA double strand break. The sequence-specific DNA binding of TALEN proteins is based on multiple 34 amino acid repeat modules, termed repeat variable di-residues (RVD), each binding specifically to one of the four nucleotides. All modules together (termed “targeting sequence”) allow the recognition of a specific DNA sequence, so that in principle any DNA sequence of interest within the genome can be targeted [22], [23]. The catalytic domain of the endonuclease FokI, being fused to this targeting sequence, is only active as a dimer. Hence, for successful FokI activity two TALEN proteins are required to bind in the right orientation and in close proximity onto the DNA double strand to induce double strand break formation. The mandatory binding of both TALENs strongly increases the targeting specificity [24]. The double strand break can be subsequently repaired by cellular mechanisms based either on homologous recombination (HR) or on non-homologous end joining (NHEJ). While NHEJ occurs during the whole cell cycle, HR is mainly restricted to the late S and G2 phase [25]. Both of these DNA repair mechanisms can be used to induce targeted mutations: HR can be exploited by introduction of foreign DNA with a strong homology to the DNA sequence surrounding the target site, which the cell uses as template to repair the double strand break [26]. If no HR template is used, the high error rate of NHEJ can be exploited to generate small random insertions/deletions until the target site is inaccessible for TALEN [27].
The successful application of TALEN and CRISPR/Cas9 in P. tricornutum has been published recently [28], [29], [30], however, a number of potential pitfalls have not yet been sufficiently addressed. Therefore, in this report, we describe how the TALEN approach can be optimized to obtain cell lines with targeted mutations at a high frequency and how to minimize the risk of potential off-target binding of TALENs. Additionally, we show that a thorough screening process is required for correctly distinguishing bi-allelic and mono-allelic knockout mutants, as well as to prove that a specific cell line is genetically homogeneous and does not contain different genotype lines. Although the CRISPR approach can be more easily adapted for a specific target site, we chose to improve the strategy of utilization of TALENs in diatoms because, based on research in other organisms [31], it offers the potential for reduced off-target effects. We developed a diatom-specific TALEN system by combining two previously published P. tricornutum transformation plasmids, pPha-T1 (Genebank ID: AF219942.1, [13]) and pPha-NR (Genebank ID: JN180663.1, [32]), and the TALEN plasmids developed by the Zhang lab for mammalian systems [33]. We adapted the Zhang protocol [33] for TALENs assembly and verification for diatoms, allowing the creation of TALENs in less than two weeks. We furthermore developed concise guidelines for all steps of this method, from target site design to screening mutated lines, which allows any molecular biology lab equipped with a particle gun or an electroporator to produce knockout mutants of P. tricornutum. In order to evaluate our TALEN system, we generated TALENs targeting the PtAureo1a gene, encoding the blue-light photoreceptor Aureochrome 1a (PtAUREO1a), because of the availability of a specific antiserum as well as the known phenotype of RNAi-silenced strains, such as lower chlorophyll a (Chl a) content per cell and a higher relative amount of xanthophyll cycle pigments [35].
Section snippets
Assembly of the TALEN plasmids
The plasmid kit used for building TALENs was a gift from Dr. Feng Zhang (Addgene, Cambridge, MA, USA; kit #1000000019) [33]. A HindIII restriction site was introduced upstream of the start codon of the TALEN plasmids using primer pair TALEN_HindIII + _for/rev (see Table SI). The P. tricornutum expression plasmids pPha-T1 (GenBank AF219942, [13]) and pPha-NR (GenBank JN180663, [32]) were modified using site-directed mutagenesis with the primer pairs PTV_BSAI1719SD_for/rev and
Generation of the TALEN constructs
In order to perform a cost-effective and easy assembly of the TALEN targeting sequence, we chose a system developed previously for mammalian systems, allowing the complete assembly and sequence verification of individual TALEN plasmids within two weeks [33]. In contrast to previous publications on P. tricornutum [28], [30] describing the expression of TALENs in a single transformation plasmid, we decided to clone the two TALEN backbones into two separate plasmids, one with a constitutive fcpA
Conclusion
An efficient TALEN system has been established for the diatom P. tricornutum, which leads to a high frequency of targeted mutation events and yields mainly genetically homogeneous cell lines. Additionally, all important steps, from target site design, TALEN construction using an easy-to-follow and publically available modular construction system, up to screening of the mutants, have been addressed to avoid the potential pitfalls for knockout generation in P. tricornutum, like potential
Acknowledgements
The authors are grateful to Doris Ballert for the help with the cultivation and transformation of P. tricornutum, Dr. Daniela Ewe for cloning of pPha-NR-Nat, as well as Simon Kienle and Florian Weeber for help in mutant screening. This work was supported by the University of Konstanz, the Graduate School Biological Sciences (GBS), the EU FP7 Marie Curie Zukunftskolleg Incoming fellowship (grant no. 291784, to BL), the Gordon and Betty Moore Foundation GBMF 4966 (grant DiaEdit to PGK) and the DFG
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both authors contributed equally.