Fully Automated One-Step Synthesis of Single-Transcript TALEN Pairs Using a Biological Foundry

Abstract

Transcription activator-like effector nuclease (TALEN) is a programmable genome editing tool with wide applications. Since TALENs perform cleavage of DNA as heterodimers, a pair of TALENs must be synthesized for each target genome locus. Conventionally, TALEN pairs are either expressed on separate vectors or synthesized separately and then subcloned to the same vector. Neither approach allows high-throughput construction of TALEN libraries for large-scale applications. Here we present a single-step assembly scheme to synthesize and express a pair of TALENs in a single-transcript format with the help of a P2A self-cleavage sequence. Furthermore, we developed a fully automated platform to custom manufacture TALENs in a versatile biological foundry. 400 pairs of TALENs can be synthesized with over 96.2% success rate at a material cost of $2.1/pair. This platform opens the door to TALEN-based genome-wide studies.

Keywords: DNA assembly; TALEN; automation; biofoundry; genetic screening; genome engineering.

Figure 1

Design and preliminary test of single-transcript TALEN synthesis. (a) Overall design. Both TALENs were transcribed as one mRNA, but they were sliced to separate proteins in translation as a P2A sequence was inserted between the open reading frames. (b) Assembly scheme. A library of all possible combinations of single and dual TALE repeats were preassembled with standardized Golden Gate linkers for each position. Thus, each TALEN monomer can target 8 to 15 nucleotides with a mix of single and dual repeats. Repeats for both monomers as well as the LR-C-terminus-Fok-I-P2A-N-terminus fragment are assembled in a single Golden Gate assembly reaction. LR: last repeat. Term.: terminus. (c) Test assembly of a single-transcript TALEN pair. 28 independent clones were picked and digested by PvuI and StuI. All had correct digestion pattern. Arrows indicate the correct digestion pattern.

Figure 2

Functional test of single-transcript TALENs. (a) Single-transcript expression of a TALEN pair. Two distinctive TALEN pairs were expressed in HEK293T cells with the single-transcript design. TALEN monomers showed visible bands on Western blot while no band for the size of the uncleaved doublet was detected. (b) Genome editing in HEK293T cells. Single-transcript TALENs (STTLN) were compared against the traditional dual plasmid TALENs (TDTLN) by targeting BRCA2 as well as ABL1 sites in HEK293T cells. T7E1 assay was performed to detect the indel introduced by TALEN cleavage and NHEJ. The STTLN transfected samples showed comparable cleavage efficiency to TDTLN transfected samples. CTRL: sample with no TALEN transfection served as negative control. (c) Disruption of an Oct4 enhancer in H1 hESC. Flow cytometry was used to quantify the GFP expression level in H1-Oct4-GFP cells. The gated population had lower than normal GFP expression. Left: control population without enhancer disruption, middle: enhancer disrupted by traditional 2-plasmid TALEN, right: enhancer disrupted by single-transcript TALEN.

Figure 3

Overview of the iBioFAB system. (a) Breakdown of unit operations. (b) Layout of iBioFAB’s hardware. iBioFAB has two robotic arms. A centralized 6-degree-of-freedom arm on a 5-m track is used to transport labware between instruments. A 3-degree-of-freedom arm moves labware inside the liquid handling station. (c) Control hierarchy of iBioFAB. Process modules are developed in the system control GUI. iScheduler is in charge of workflow level control. Script Generator generates pipetting routes for the liquid handling GUI. Process modules can be quickly recombined to compose different workflows.

Figure 4

Fully automated synthesis of TALEN libraries. (a) General workflow for the DNA assembly pipeline based on the Golden Gate method. Script Generator converted project design ideas, such as permutations of DNA parts, to assembly designs with appropriate extensions and further robotic commands for pipetting the stock plasmids to DNA mixes. In Golden Gate reactions, Type IIs restriction enzymes such as BsaI generated a set of standard precharacterized 4-bp single strained ends as linkers. The corresponding linkers annealed and were ligated by T4 ligase. (b) Process flow diagram for the build step. Unit operations employed were marked in blue. (c) Verification of single-transcript TALENs synthesized in high throughput. 94 samples were randomly selected from the 192 TALEN pairs synthesized in the full batch test. Each plasmid sample encoding a pair of TALENs was extracted from a polyclonal E. coli cell culture and restriction digested. The fragment sizes were analyzed by capillary electrophoresis. The digestion pattern was simulated. (d) Summary of efficiency, throughput, and cost.