TAL effector nucleases induce mutations at a pre-selected location in the genome of primary barley transformants
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- Wendt, T., Holm, P.B., Starker, C.G. et al. Plant Mol Biol (2013) 83: 279. doi:10.1007/s11103-013-0078-4
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Transcription activator-like effector nucleases (TALENs) enable targeted mutagenesis in a variety of organisms. The primary advantage of TALENs over other sequence-specific nucleases, namely zinc finger nucleases and meganucleases, lies in their ease of assembly, reliability of function, and their broad targeting range. Here we report the assembly of several TALENs for a specific genomic locus in barley. The cleavage activity of individual TALENs was first tested in vivo using a yeast-based, single-strand annealing assay. The most efficient TALEN was then selected for barley transformation. Analysis of the resulting transformants showed that TALEN-induced double strand breaks led to the introduction of short deletions at the target site. Additional analysis revealed that each barley transformant contained a range of different mutations, indicating that mutations occurred independently in different cells.
KeywordsTAL effector nucleasesTargeted mutagenesisHordeum vulgareCereal transformationTALEN
Rapidly advancing DNA sequencing technologies have led to the availability of sequence information that provides a significant asset for future breeding efforts. Recently, the draft assembly was released of the genome of the commercially important cereal, barley (Hordeum vulgare L.) (The International Barley Genome Sequencing Consortium 2012). However, utilizing this sequence information to further improve breeding material remains a major challenge.
Barley is considered a model plant for species within the Triticeae tribe (i.e. wheat and rye) since it is a well-characterized, self-pollinating diploid species. Further, a number of molecular and genetic techniques are available, including an Agrobacterium-mediated transformation system (Schulte et al. 2009; Tingay et al. 1997). For many of the gene sequences recently identified in barley, with corresponding sequences identified in wheat and rye, a function is still not fully assigned. Gene inactivation is a powerful tool for investigating and validating gene function. In species with high transformation efficiencies, inactivation of specific genes is often accomplished by random insertional mutagenesis using transposons or T-DNAs followed by screening of the mutant populations to identify desired mutations (Jeon et al. 2000). Antisense or RNAi techniques may also be implemented to reduce or eliminate gene expression (Matzke et al. 2001). Alternatively, mutations can be randomly induced by chemical or physical mutagens. However, for the species within the Triticeae tribe, these methods are laborious, due to the complexity and size of the genomes and comparatively modest transformation efficiencies. Furthermore, conventional mutagenesis techniques have the disadvantage in that DNA damage is imposed at random positions in the genome, and a plethora of modifications are generated such as substitutions, insertions, deletions, inversions and translocations (Sikora et al. 2011).
Recently, technologies have been developed to enable precise alteration of gene sequences at specific sites in a genome. These technologies are based on DNA sequence-specific binding proteins with nuclease activity, such as meganucleases, zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) (Epinat et al. 2003; Puchta and Hohn 2010; Bogdanove and Voytas 2011). These nucleases can be designed to (1) selectively bind to specific locations in a genome and (2) create double strand breaks (DSBs) at the binding site. Another technology was recently developed that utilizes short RNAs to guide a nuclease (Cas9) to complementary DNA targets where site-specific DSBs are introduced (Jinek et al. 2012). This system, named Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas is, however, not yet optimized for expression and use in plants. The DSBs that are induced by these technologies are repaired by the cell’s native machinery. The two cellular DNA repair mechanisms are non-homologous end joining (NHEJ) and homologous recombination (HR). The primary pathway, NHEJ, is known to result in mutations in the form of short deletions, insertions and/or substitutions.
While TALENs were only recently developed, they have already proven effective in inducing site-specific mutations at high efficiencies in various organisms, including the plant species Arabidopsis, tobacco, Brachypodium and rice (Bedell et al. 2012; Cermak et al. 2011; Dahlem et al. 2012; Li et al. 2012; Shan et al. 2013; Tong et al. 2012; Zhang et al. 2013). Even before the development of TALENs, both meganucleases and ZFNs were shown to successfully induce mutations at pre-selected genomic locations; however, the expertise required for their engineering and the cost involved has prevented their widespread use. The main advantages of TALENs are their specificity, ease of assembly and expansive targeting range. Today, TALENs can be designed for any location in any genome, whereas the targeting range of ZFNs and meganucleases is still constrained.
The potential of the TALEN technology for crop improvement efforts was recently demonstrated by Li et al. (2012). This study showed high frequency targeted mutagenesis in rice. Mutations at the target site (the promoter of a bacterial blight susceptibility gene) led to the recovery of pathogen resistant rice plants. The TALENs were introduced into rice by Agrobacterium-mediated transformation; however, since the T-DNA encoding the TALENs is only rarely linked to the target site, Li et al. (2012) were able to identify progeny with mutations that lacked the TALEN T-DNA. This allowed for the propagation of lines without transgenes.
At present, it takes eight to nine months to generate mature T0 barley plants using conventional Agrobacterium-mediated transformation of immature embryos. Implementing and validating new technologies based on transformation is therefore slow. Here we report current results obtained in primary barley transformants using the TALEN technology. We chose as a target for our TALENs a site in the promoter of a barley phytase gene of the purple acid phosphatase group named HvPAPhy_a (Holme et al. 2012). This gene was previously shown to account for most of the phytase activity in the developing barley grain (Dionisio et al. 2011). Within this promoter we targeted a region that contains a group of regulatory motifs involved in the expression of genes during grain development (Madsen et al. 2013). Ultimately, the goal of this study is to validate the importance of these motifs by investigating whether mutations in these sequences result in altered phytase expression and activity in the developing barley grain. In this study we show that it is possible to identify a high percentage of primary transformants with a variety of deletions at this pre-selected location in the barley genome, indicating the potential of TALENs as a powerful tool for barley research and breeding.
Materials and methods
TALEN assembly and yeast-based cleavage assay
For TALEN design, the genomic sequence of the HvPAPhy_a promotor (Holme et al. 2012) was analyzed using the online software tool TALE-NT1.0 (Doyle et al. 2012; Cermak et al. 2011). Three TALEN recognition sites (Online Resources 1) were chosen based on their proximity to the target region. The TALENs were assembled according to their corresponding repeat variable diresidues (RVDs) (Online Resources 1) using the GoldenGate cloning strategy (Cermak et al. 2011; Christian et al. 2010; Addgene http://www.addgene.org/TALeffector/goldengate/voytas/). Assembled TALEN arrays were cloned into yeast expression vectors pTAL3/pTAL4. The homologue of the genomic target sequence was cloned into the SpeI/BglII (NEB) site of pCP5 (Zhang et al. 2010). Average cleavage frequency of each TALEN was estimated using a yeast-based assay (Townsend et al. 2009; Gietz et al. 1997). Cleavage activity was normalized against a ZFN control (Zif268) and tested on specific and non-specific targets.
Vector construction for plant transformation
The spring cultivar Golden Promise was grown in growth cabinets at 15 °C during the day and 10 °C at night with a 16 h light period and a light intensity of 350 μ E m−2 s−1. Immature embryos isolated 12–14 days after pollination were used for Agrobacterium-mediated transformation following the procedure of Holme et al. (2012). Callus was induced on infected immature embryos on hygromycin-containing medium, and plantlets resistant to hygromycin were regenerated.
Plant material for molecular analysis was collected from the second leaf of antibiotic resistant primary transformants. Genomic DNA was isolated using The FastDNA™ Kit (MP Biomedicals). Pre-digestion of DNA with DrdI (NEB) was carried out for 2 h at 37 °C in 10 μl total volume (300–500 ng plant DNA, 1X NEB4, 1X BSA, 1U DrdI). PCR on 30–50 ng pre-digested DNA with the forward primer: 5′-taatctggccgaaccttgtta-3′; and reverse primer: 5′-agaatcaatgccttgccatc-3′ was used to amplify the TALEN target site. Phusion High-Fidelity DNA Polymerase (Thermo Scientific) was used for these PCR-reactions according to the manufacturer’s instructions. The amplified PCR products were digested with DrdI for 1 h at 37 °C. Digestion profiles were analyzed on 1 % agarose gels. Undigested bands were gel purified (GenElute™ Gel Extraction Kit, Sigma Aldrich) and cloned using the TOPO Zeroblunt® cloning kit (Invitrogen). Cloned products were sequenced and analyzed by alignment in ApE (A plasmid Editor v2.0.36).
Results and discussion
It was recently reported that TALEN cleavage efficiency can be increased when the TAL effector backbone is truncated at the N- and C-termini (Miller et al. 2007; Zhang et al. 2013). We therefore performed experiments with the NΔ288/CΔ231 scaffold (T-DNA size of 11.8 Kb) and the NΔ152/CΔ63 scaffold (T-DNA size of 9.5 Kb) (Fig. 1). A total of 658 immature embryos were infected with the NΔ288/CΔ231 scaffold. For this treatment 89 (13.5 %) antibiotic resistant transformants were recovered. An increase in transformation efficiency was observed when the NΔ152/CΔ63 scaffold was used with the same TAL effector array (i.e. DNA binding specificity of the two TALENs was the same). Here, a total of 473 immature embryos were infected, and from these, 105 (22.2 %) developed antibiotic resistant transformants. The observed increase could be a direct effect of the reduced T-DNA size.
To analyze whether one particular mutation of the initially transformed cell was carried through mitotic cell divisions of one individual plant, we independently sequenced several samples from the same transformant. We found that several different mutations were present in a single plant (Fig. 4c). Therefore independent mutations must have occurred in some cells, rather than all cells being derived from a single mutation event. The occurrence of TALEN-induced mosaicism was also reported in zebrafish (Bedell et al. 2012) that were treated with TALEN mRNA. A potential advantage of a variety of mutations in T0 plantlets is that any of these mutations may be transmitted through the gametes and inherited in the next generation. Therefore, a single transgenic plant may give rise to T1 seedlings each carrying different mutations. This would greatly reduce the time and effort needed to generate mutant libraries from primary transformants.
In this study, we were able to show that large TALEN constructs can be expressed in barley. An average of one out of four antibiotic resistant primary transformants showed the presence of mutations, indicating that it is not necessary to generate large numbers of transformants for analysis. The identification of barley plantlets carrying TALEN-induced mutations was increased using the NΔ152/CΔ63 scaffold, a trend that was also reported for tobacco protoplasts (Zhang et al. 2013). Our results complement earlier findings in plants (Arabidopsis, tobacco, Brachypodium and rice) and report similar mutation patterns as seen in other organisms. Therefore these results should encourage the research community to implement this technology in species with relatively low transformation efficiencies such as barley and other Triticeae species.
This project is funded by the Danish Ministry of Food, Agriculture and Fisheries (3304-FVFP-09-B-006) and a grant from the US National Science Foundation (DBI 0923827).