TAL effector nucleases induce mutations at a pre-selected location in the genome of primary barley transformants

Abstract

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.

Fig. 1

Schematic overview of TALEN plant expression vectors. Two constructs (N∆288/C∆231 and N∆152/C∆63) with the same TALEN array were prepared and cloned into the destination vector pH2GW7 (Karimi et al. 2002). Both constructs contain the plant selection gene hygromycin (hygR). TAL arrays are separated by a skipping peptide (T2A) for individual expression from the same transcript generated by the constitutive 35S promoter. Cleavage domains are two heterodimeric FokI subunits (Doyon et al. 2011). T35S, the 35S transcriptional terminator

Fig. 2

Yeast-based assay to determine TALEN cleavage frequency and specificity. Each TALEN was tested against the homologue of its specific genomic target site (sp. Target) and against an unspecific binding site (unsp. Target) close to its genomic location. Cleavage frequency was estimated as beta-galactosidase units resulting from the reconstitution of a disrupted lacZ gene. All values were normalized against a Zif268 ZFN control. Error bars depict Standard Deviation (n = 3)

Fig. 3

Overview of an enrichment assay to detect mutations at the target site in the HvPAPhy_a promoter. a TALENs were designed so that the spacer region in between the TALEN binding sites has a restriction site (DrdI). Specific primers (arrows) amplify a 1,051 bp fragment, which can be cut into two fragments of 529 and 522 bp with DrdI. b For the enrichment assay, genomic DNA was pre-digested with DrdI (1). The digested DNA was used for a specific PCR reaction that amplifies the target fragment (2) in antibiotic resistant plants (1–6). As controls, an untransformed sample (wt) and a no template control (H₂O) were included. The PCR product was then digested with DrdI (3). Fragments of the final digestion were analyzed on 1 % agarose gels for three types of patterns: (i) fragments of 529 and 522 bp = wild type only (B3, lanes 7, 8); (ii) fragments of 1,051, 529 and 522 bp = wildtype and mutated (B3, lanes 10, 11, 12); (iii) a fragment of 1,051 bp = mutated only (B3, lane 9)

Fig. 4

Sequence alignments of the target region (WT) and the corresponding region in genomic DNA of T₀ plantlets. Samples represent cloned PCR products of individual T₀ plantlets derived from the enrichment assay. Plants were transformed with either a N∆288/C∆231 scaffold or b N∆152/C∆63 scaffold. c Several independent clones of the same PCR product from one indivdual T₀ plant (Line 12; N∆152/C∆63 scaffold) were individually sequenced and aligned to the target region. Underlined sequences represent the TALEN binding sites. The number of deletions for each sample is indicated on the right site