ULtiMATE system for rapid assembly of customized TAL effectors

Abstract

Engineered TAL-effector nucleases (TALENs) and TALE-based constructs have become powerful tools for eukaryotic genome editing. Although many methods have been reported, it remains a challenge for the assembly of designer-based TALE repeats in a fast, precise and cost-effective manner. We present an ULtiMATE (USER-based Ligation Mediated Assembly of TAL Effector) system for speedy and accurate assembly of customized TALE constructs. This method takes advantage of uracil-specific excision reagent (USER) to create multiple distinct sticky ends between any neighboring DNA fragments for specific ligation. With pre-assembled templates, multiple TALE DNA-binding domains could be efficiently assembled in order within hours with minimal manual operation. This system has been demonstrated to produce both functional TALENs for effective gene knockout and TALE-mediated gene-specific transcription activation (TALE-TA). The feature of both ease-of-operation and high efficiency of ULtiMATE system makes it not only an ideal method for biologic labs, but also an approach well suited for large-scale assembly of TALENs and any other TALE-based constructions.

Figure 1. Customized TALE construction by ULtiMATE system.

(A) Flowchart of the cloning process of a 17.5-mer TALE targeting ATCTTTAAACCCCTAGTC. The selection of templates and primers are based on the archive of 64 pre-assembled trimers and the 40-primer pool (Tables S1 and S2). The PCR amplification of trimers can be finished within 1.5 hrs. Without the need of purification, the PCR products of all reactions are mixed for subsequent USER^TM enzyme digestion and ligation sequentially in the same tube. The ligated fragments are gel-purified before mixed with TALE cloning backbone (Data S1B) for the cycle of BsmBI digestion and ligation, followed by the bacterial transformation (~ 1.5 hrs). The candidate clones are isolated based on colony PCR results, and verified by sequencing analysis. (B) PCR amplification of the trimers indicated by electrophoresis. Marker 1 is 100-bp DNA ladder (same below). (C) USER-mediated ligation indicated by electrophoresis. Red arrows refer to ligated DNA bands with correct sizes, ~1.8 kb. (D) Colony PCR of six randomly picked clones after transformation. Marker 2 is 1-kb DNA ladder. The DNA bands with correct size (~1.8 kb) are indicated by red arrows.

Figure 2. Effects of TALE-TAs in human cell lines.

(A) Design and structure of a representative luciferase reporter, Triple A-Luc, and its corresponding TALE transactivator, TALE-(NI)_7,8,9. The reporter contains an artificial TALE-binding sequence (CTGGCCAAATACGTA) upstream a mini CMV promoter, followed by the Firefly luciferase gene. TALE-(NI)_7,8,9 carries the Triple A binding TALE, fused with VP64 viral activation domain. The TALE-binding sequences for Triple-A, -C, -G and -T reporters differ only in 3 consecutive bases in the middle. (B) The binding activity of each TALE-TAs is determined by measuring the relative luciferase units (RLU) for their corresponding reporter activity after normalization with a co-transfected Renilla expressing vector pRL-TK. Error bars indicate standard deviations of four replicates.

Figure 3. Examples of disruption of genes in human cell lines by ULtiMATE-engineered TALENs.

(A, D and G) Partial sequences of HBEGF, ANTXR1, and LRP1 genes in genome containing TALENs binding regions (overlined for TALEN^L and underlined for TALEN^R). Restriction enzyme cutting sites are highlighted in yellow. (B, E and H) Measurement of indel rates in TALENs-treated HeLa and HEK293T cells by restriction enzyme digestion. The uncleaved bands indicate potential indels. Both wild type and cells treated by TALENs targeting ANTXR2 gene are used as controls. The percentage of uncleaved band (indicated by red arrow) was measured using ImageJ (http://rsbweb.nih.gov/ij/). (C, F and I) Sequencing analysis of mutated alleles from 4-6 randomly selected TALENs clones (in HeLa cells). The TALENs binding sites (underlined) and restriction enzyme cutting sites (in yellow) are highlighted. Dashes and red letters indicate deletions and insertions, respectively.