A transcription activator-like effector toolbox for genome engineering

Abstract

Transcription activator-like effectors (TALEs) are a class of naturally occurring DNA-binding proteins found in the plant pathogen Xanthomonas sp. The DNA-binding domain of each TALE consists of tandem 34-amino acid repeat modules that can be rearranged according to a simple cipher to target new DNA sequences. Customized TALEs can be used for a wide variety of genome engineering applications, including transcriptional modulation and genome editing. Here we describe a toolbox for rapid construction of custom TALE transcription factors (TALE-TFs) and nucleases (TALENs) using a hierarchical ligation procedure. This toolbox facilitates affordable and rapid construction of custom TALE-TFs and TALENs within 1 week and can be easily scaled up to construct TALEs for multiple targets in parallel. We also provide details for testing the activity in mammalian cells of custom TALE-TFs and TALENs using quantitative reverse-transcription PCR and Surveyor nuclease, respectively. The TALE toolbox described here will enable a broad range of biological applications.

Figure 1. A TALE toolbox for genome engineering

(a) Natural structure of TALEs derived from Xanthomonas sp. Each DNA binding module consists of 34 amino acids, where the repeat variable diresidues (RVDs) in the 12^th and 13^th amino acid positions of each repeat specify the DNA base being targeted according to the cipher NG = T, HD = C, NI = A, and NN = G or A. The DNA binding modules are flanked by non-repetitive amino and carboxyl termini, which carry the translocation, nuclear localization (NLS), and transcription activation (AD) domains. A cryptic signal within the amino terminus specifies a thymine as the first base of the target site. (b) The TALE toolbox allows rapid and inexpensive construction of custom TALE-TFs and TALENs. The kit consists of 12 plasmids in total: 4 monomer plasmids to be used as templates for PCR amplification, 4 TALE-TF and 4 TALEN cloning backbones corresponding to 4 different bases targeted by the 0.5 repeat. CMV: cytomegalovirus promoter; N-term: non-repetitive amino terminus from the Hax3 TALE; C-term: non-repetitive carboxyl terminus from the Hax3 TALE; BsaI: type IIs restriction sites used for the insertion of custom TALE DNA binding domains; ccdB+CmR: negative selection cassette containing the ccdB negative selection gene and chloramphenicol resistance gene; NLS: nuclear localization signal; VP64: synthetic transcriptional activator derived from VP16 protein of herpes simplex virus; 2A: 2A self-cleavage linker; EGFP: enhanced green fluorescent protein; polyA signal: polyadenylation signal; FokI: catalytic domain from the FokI endonuclease. (c) TALEs can be used to generate custom transcription factors (TALE-TFs) and modulate the transcription of endogenous genes from the genome. This schematic shows a TALE-TF designed to target the SOX2 locus in the human genome. The SOX2 TALE-TF recognizes the sense strand of the SOX2 proximal promoter, and the recognition site begins with T. The TALE DNA-binding domain is fused to the synthetic VP64 transcriptional activator, which recruits RNA polymerase and other factors needed to initiate transcription. (d) TALE nucleases (TALENs) can be used to generate site-specific double strand breaks to facilitate genome editing through non-homologous repair or homology-directed repair. This schematic shows a pair of TALENs designed to target the AAVS1 locus in the human genome. Two TALENs target a pair of binding sites flanking a 16bp spacer. The left and right TALENs recognize the top and bottom strands of the target sites respectively. Each TALE DNA-binding domain is fused to the catalytic domain of FokI endonuclease; when FokI dimerizes, it cuts the DNA in the region between the left and right TALEN binding sites.

Figure 2. Timeline for the construction of TALE-TFs and TALENs

Steps for the construction and functional testing of TALE-TFs and TALENs are outlined. TALEs can be constructed and sequence verified in 5 days following a series of ligation and amplification steps. During the construction phase, samples can be stored at −20°C at the end of each step and continued at a later date. After TALE construction, functional validation via qRT-PCR (for TALE-TFs) and Surveyor nuclease assay (for TALENs) can be completed in 2–3 days.

Figure 3. Construction of TALE DNA binding domains using hierarchical ligation assembly

Schematic of the construction process for a custom TALE containing a 18-mer tandem repeat DNA binding domain. Stage 1: specific primers are used to amplify each monomer and add the appropriate ligation adapters (Procedure Steps 1–9). Stage 2: hexameric tandem repeats (1—6, 7—12, and 13—18) are assembled first using Golden Gate digestion-ligation. The 5′ ends of monomers 1, 7, and 13 and the 3′ ends of monomers 6, 12, and 18 are designed so that each tandem hexamer assembles into an intact circle (Procedure Steps 10–15). Stage 3: the Golden Gate reaction is treated with an exonuclease to remove all linear DNA, leaving only the properly assembled tandem hexamer (Procedure Steps 16–17). Stage 4: each tandem hexamer is amplified individually using PCR and purified (Procedure Steps 18–25). Stage 5: tandem hexamers corresponding to 1—6, 7—12, and 13—18 are ligated into the appropriate TALE-TF or TALEN cloning backbone using Golden Gate cut-ligation (Procedure Steps 26–28). Stage 6: The assembled TALE-TF or TALEN is transformed into competent cells and successful clones are isolated and sequence verified (Procedure Steps 29–38).

Figure 4. PCR plate setup used to generate a plate of monomers for constructing custom 18-mer TALE DNA binding domains

One 96-well plate can be used to carry out 72 reactions (18 for each monomer template). The position of each monomer and the primers used for the position is indicated in the well. Color coding in the well indicates the monomer used as the PCR template. Typically, 2–4 plates of 100 ul PCR reactions are pooled together and purified to generate a monomer library of sufficient quantity for production of many TALEs. During TALE construction, the corresponding monomer for each DNA base in the 18 bp target sequence can be easily picked from the plate.

Figure 5. Example gel results from the TALE construction procedure

(a) Lanes 1—6: products from the monomer PCR reaction (Stage 1 in Figure 3) after purification and gel normalization (Procedure Steps 8–9). The molar concentrations of samples shown on this gel have been normalized so that equal moles of monomers are mixed for downstream steps. Monomers 1 and 6 are slightly longer than monomers 2, 3, 4, and 5 due to the addition of sequences used for circularization. Lane 7: result of the hexamer Golden Gate cut-ligation (Procedure Step 15). A series of bands with size ~700 bp and lower can be seen. Successful hexamer Golden Gate assembly should show a band ~700 bp (as indicated by arrow). Lane 8: hexamer assembly after PlasmidSafe exonuclease treatment (Procedure Step 17). Typically the amount of circular DNA remaining is difficult to visualize by gel. Lane 9: result of hexamer amplification (Procedure Step 20). A ~700 bp band should be clearly visible. The hexamer gel band should be gel-purified to remove shorter DNA fragments. (b) Properly assembled TALE-TFs and TALENs can be verified using bacterial colony PCR (2175 bp band, lane 1) (Procedure Step 35) and restriction digest with AfeI (2118 bp band for correctly assembled 18-mer in either backbone; other bands for TALE-TF are 165 bp, 3435 bp, 3544 bp; other bands for TALEN are 165 bp, 2803 bp, 3236 bp; digest shown is for TALE-TF backbone vector, lane 2) (Procedure Step 35).

Figure 6. Examples of TALE-TF and TALEN activity in 293FT cells

(a) Schematic of the Surveyor nuclease assay used to determine TALEN cleavage efficiency. First, genomic PCR is used to amplify the TALEN target region from a heterogeneous population of TALEN-modified and unmodified cells, and the gPCR products are re-annealed slowly to generate heteroduplexes. The re-annealed heteroduplexes are cleaved by Surveyor nuclease while homoduplexes are left intact. TALEN cleavage efficiency is calculated based on the fraction of cleaved DNA. (b) Gel showing the Surveyor nuclease result from the AAVS1 TALEN pair (from Fig. 1d). Lanes 1—4: controls from un-transfected (N.T.) cells and cells transfected with a plasmid carrying GFP (Mock), AAVS1 left TALEN only (L), and AAVS1 right TALEN only (R). Lanes 5—7: cells transfected with AAVS1 Left and Right TALENs (L+R) for 24, 48, and 72 hours. The two lower bands indicated by the arrows are Surveyor-cleaved DNA products. (c) 293FT cells transfected with the SOX2 TALE-TF (from Fig. 1c) exhibited a 5 fold increase in the amount of SOX2 mRNA compared with mock transfected cells. Error bars indicate s.e.m.; n = 3. *** indicates P < 0.005. Panel c was modified with permission from Nature Biotechnology (Nature Biotechnology (c) 2011, Macmillian Publishers Ltd.).