Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting

Abstract

TALENs are important new tools for genome engineering. Fusions of transcription activator-like (TAL) effectors of plant pathogenic Xanthomonas spp. to the FokI nuclease, TALENs bind and cleave DNA in pairs. Binding specificity is determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors. We present a method and reagents for efficiently assembling TALEN constructs with custom repeat arrays. We also describe design guidelines based on naturally occurring TAL effectors and their binding sites. Using software that applies these guidelines, in nine genes from plants, animals and protists, we found candidate cleavage sites on average every 35 bp. Each of 15 sites selected from this set was cleaved in a yeast-based assay with TALEN pairs constructed with our reagents. We used two of the TALEN pairs to mutate HPRT1 in human cells and ADH1 in Arabidopsis thaliana protoplasts. Our reagents include a plasmid construct for making custom TAL effectors and one for TAL effector fusions to additional proteins of interest. Using the former, we constructed de novo a functional analog of AvrHah1 of Xanthomonas gardneri. The complete plasmid set is available through the non-profit repository AddGene and a web-based version of our software is freely accessible online.

Figure 1.

TAL effector and TALEN structure. (a) Structure of a naturally occurring TAL effector. A consensus repeat sequence is shown with the repeat-variable di-residue (RVD) underlined. The sequence of RVDs determines the target nucleotide sequence. The four most common RVDs, on which our designs and plasmids are based, are shown with their most frequently associated nucleotide. Some evidence suggests that the less common RVD NK (not displayed) has greater specificity for G than NN does and for that reason our plasmid set also includes NK modules. (b) Structure of a TALEN. Two monomeric TALENs are required to bind the target site to enable FokI to dimerize and cleave DNA. NLS, nuclear localization signal(s); AD, transcriptional activation domain; B, BamHI; S, SphI.

Figure 2.

Golden Gate assembly of custom TAL effector and TALEN constructs using module, array, last repeat and backbone plasmids. By using the type IIS restriction endonucleases BsaI and Esp3I, modules containing the desired RVDs can be released with unique cohesive ends for ordered, single-reaction assembly into array plasmids in a first step, and those arrays subsequently released and assembled in order in a second step into a backbone plasmid to create full length constructs with custom repeat arrays (see text for details). NLS, nuclear localization signal(s); AD, transcriptional activation domain; tet, tetracycline resistance; spec, spectinomycin resistance; amp, ampicillin resistance; attL1 and attL2, recombination sites for Gateway cloning; B, BamHI, and S, SphI, useful for subcloning custom repeat arrays. Unique restriction enzyme sites flanking the coding sequences, useful for subcloning the entire constructs into other vectors, are not shown but can be found in the sequence files (Supplementary Data).

Figure 3.

TALEN or TAL effector construct assembly timeline.

Figure 4.

Nucleotide and RVD frequencies at the termini of 20 target and TAL effector pairs. RVDs that have a frequency of ≥20% at one or more of the positions are shown. ‘XX’ represents all other RVDs.

Figure 5.

Activity of 15 custom TALEN pairs targeting diverse sequences in a reporter-based yeast assay. TALENS were targeted to gene sequences from the indicated organisms and to GFP and eGFP using the software and constructed using the Golden Gate method and plasmids described in the text. Activity was measured in a yeast-based assay in which cleavage and recombination reconstitutes a functional lacZ gene (see text for details). Activity was normalized to a Zif268 ZFN positive control. Activity of target-only controls for each is plotted above the target-plus-TALEN values; in each case the activity was undetectable. Error bars denote s.d.; n = 3.

Figure 6.

Site-directed mutagenesis in human embryonic kidney cells and Arabidopsis protoplasts using custom TALENs. TALENs targeted to the human HPRT1 gene (pair HPRT1 B in Figure 5) and the Arabidopsis ADH1 gene (Figure 5) were transiently expressed in human embryonic kidney cells and in Arabidopsis protoplasts, respectively and the targets subsequently amplified and sequenced (see text for details). Prior to amplification, genomic DNA was digested with a restriction endonuclease having a site present in the TALEN target site to reduce amplification of wild-type sequences and enrich the amplicon pool for mutated ones. Results for HPRT1 are shown in (a) and ADH1 in (b). For each, the schematic at the top shows the chromosomal locus, short arrows designate primers used for PCR amplification following TALEN transient expression, sequence of the wild-type gene (top line) and unique mutated alleles obtained are shown below, binding sites for the TALEN monomers are underlined and the coincident restriction endonuclease site is indicated.

Figure 7.

Activity of an AvrHah1 analog created using the Golden Gate method and our plasmid set. Shown are leaves of pepper varieties ECW30R, carrying the Bs3 resistance gene and ECW, lacking it, 48 h following spot-infiltration with suspensions of X. campestris pv. vesicatoria strain 85–10 transformed to deliver (1) Tal1c (the effector used to make the backbone plasmids in this study), (2) native AvrHah1 or (3) an AvrHah1 analog encoded by a construct made using the Golden Gate method and our plasmid set. Leaves were cleared with ethanol to reveal the accumulation of phenolic compounds, visible as dark stained areas, indicative of the hypersensitive reaction induced by TAL effector driven transcriptional activation of Bs3.