A ligation-independent cloning technique for high-throughput assembly of transcription activator–like effector genes

Abstract

Transcription activator–like (TAL) effector proteins derived from Xanthomonas species have emerged as versatile scaffolds for engineering DNA-binding proteins of user-defined specificity and functionality. Here we describe a rapid, simple, ligation-independent cloning (LIC) technique for synthesis of TAL effector genes. Our approach is based on a library of DNA constructs encoding individual TAL effector repeat unit combinations that can be processed to contain long, unique single-stranded DNA overhangs suitable for LIC. Assembly of TAL effector arrays requires only the combinatorial mixing of fluids and has exceptional fidelity. TAL effector nucleases (TALENs) produced by this method had high genome-editing activity at endogenous loci in HEK 293T cells (64% were active). To maximize throughput, we generated a comprehensive 5-mer TAL effector repeat unit fragment library that allows automated assembly of >600 TALEN genes in a single day. Given its simplicity, throughput and fidelity, LIC assembly will permit the generation of TAL effector gene libraries for large-scale functional genomics studies.

Figure 1. The LIC assembly approach to generating TALEN genes

(a) Architecture of the previously published Δ152/+63-AvrBs3-like TALEN that harbors a truncated N terminus (Δ152), an invariant recognition domain for thymine followed by a stretch of several repeat units, and a truncated and optimized C terminus (+63) fused to a FokI domain. One repeat unit is highlighted indicating the 34 amino acids and the RVDs in position 12 and 13 that were used. At right, a model of a minimal cloning unit consisting of two consecutive repeat units flanked by ID1 and ID2. (b) The library of the 64 possible 2-mer combinations with ID 1/2, 2/3, 3/4 and 4/1. (c,d) First level assembly: three 2-mers containing the target sequences are picked with alternating ID combinations (c); three 2-mers are assembled to generate a complex of six tandem repeats (6-mer) into a level 1 backbone that encodes for kanamycin resistance (kanaR) (d). (e,f) Second level assembly: assembly of a full TAL effector gene is achieved upon combining three 6-mers (e), and a mammalian expression vector containing the Δ152/+63-AvrBs3-like TAL effector backbone with a C-terminal FokI nuclease domain (f). Additional features of the mammalian expression vector (level 2 vector) are highlighted (pCMV, cytomegalovirus-promoter, ampR, bacterial resistance gene for ampicillin; NLS, nuclear localization signal; ATG, start codon; STOP, stop codon; T7, promoter sequence for T7 RNA polymerase; XhoI, XbaI, NotI, recognition sequences of the restriction enzymes XhoI, XbaI and NotI, respectively). To prevent undigested fragments from being propagated to the next level of assembly, we switched antibiotic resistance at each level.

Figure 2. LIC assembly and validation of a TALEN targeting STAT6

(a) A schematic view of the human STAT6 gene locus with the targeting site of TALEN pair 72 (yellow letters indicate the intronic sequence portion). (b) The T72L and T72R TALEN pair tested for genome editing activity in HEK 293T cells using the T7EI assay. The left panel provides a sketch of the PCR amplicon used for the assay, whereas the arrows indicate the primers used and the vertical line indicates the expected location of the nuclease activity. The right panel depicts an agarose gel of one representative result. The white numbers indicate quantified mutation frequencies. u, uncut DNA.

Figure 3. Large-scale assembly of TALENs using LIC

(a) The workflow of the 32 TALEN pair assembly. (b) Thirty-two TALEN pairs tested for functional activity in HEK 293T cells using the T7EI assay. Presented are the differences in measured mutation frequency between the TALEN-treated cells and untransfected control cells. BB, backbone.

Figure 4. Evaluation of targeting activity dependence on spacer length and RVD composition

(a) Of all TALEN pairs tested, pairs with a spacer length of 12–19 bp were plotted for genome editing activity versus spacer length. n.t., not tested (b) The relative RVD composition of the 42 tested TALEN pairs (left y-axis) alongside their respective targeting efficiencies (right y-axis).

Figure 5. A 5-mer library for LIC assembly of TALENs

The schematic of the assembly strategy of the 5-mer fragment library. (a) To obtain all possible 5-mer fragments of one ID combination, 16 2-mer fragments in two different ID combinations and 4 1-mer fragments are assembled to give rise to 1,024 5-mer fragments. (b) The 5-mer fragments are then cloned into level 1 backbones available in three different ID combinations to obtain 3 × 1,024 5-mer fragments. (c) The arrayed 5-mer fragment library is digested and chewed back in 384-well plates. A liquid-handling robot picks and mixes the appropriate 5-mer fragments with their appropriate level 2 backbone plasmid. After transformation, bacteria are grown under limiting dilution conditions to obtain monoclonal liquid cultures. A correctly assembled TAL effector construct is identified using a restriction digest and an optional sequencing reaction.

Figure 6. On target activity of 15.5-RVD LIC TALENs

(a) A schematic view of the human STAT6 gene locus with the targeting sites of TALEN pairs T499, T500 and T501 (yellow letters indicate the intronic sequence portion). (b) The TALEN pairs were tested for genome editing activity in HEK 293T cells using the T7EI assay. The panel depicts an agarose gel of one representative result out of two. The white numbers indicate quantified mutation frequencies. *, nonspecific PCR product.