Transcription activator like effector (TALE)-directed piggyBac transposition in human cells

Abstract

Insertional therapies have shown great potential for combating genetic disease and safer methods would undoubtedly broaden the variety of possible illness that can be treated. A major challenge that remains is reducing the risk of insertional mutagenesis due to random insertion by both viral and non-viral vectors. Targetable nucleases are capable of inducing double-stranded breaks to enhance homologous recombination for the introduction of transgenes at specific sequences. However, off-target DNA cleavages at unknown sites can lead to mutations that are difficult to detect. Alternatively, the piggyBac transposase is able perform all of the steps required for integration; therefore, cells confirmed to contain a single copy of a targeted transposon, for which its location is known, are likely to be devoid of aberrant genomic modifications. We aimed to retarget transposon insertions by comparing a series of novel hyperactive piggyBac constructs tethered to a custom transcription activator like effector DNA-binding domain designed to bind the first intron of the human CCR5 gene. Multiple targeting strategies were evaluated using combinations of both plasmid-DNA and transposase-protein relocalization to the target sequence. We demonstrated user-defined directed transposition to the CCR5 genomic safe harbor and isolated single-copy clones harboring targeted integrations.

Figure 1.

PB targeting plasmids. The TREs flank the PB transposon. The 3′TRE resides within an introduced intron in the PB gene leading to inactivation of the transposase on excision of the transposon. The CAG promoter drives expression of the PB targeting proteins and the IRES allows for dual expression of two proteins by the promoter. The UAS and R1 recognition sequences for the Gal4 and TALR1 DBDs were engineered into the plasmid backbones. The transgene can be Gateway recombined between the attR sites. (A) hG3-TALC1 (B) hGT1-TALC1 (C) hGT2-TALC2 (D) hGT3-TALC1 (E) hGR1T1-TALC1.

Figure 2.

Schematic for various PB targeting strategies. For each strategy, all components are encoded on a single plasmid, and most components have been omitted for simplification purposes from plasmids depicted in this figure. (A) hG3-TALC1 encodes a hyperactive PB transposase covalently linked to a TALE designed to bind a specific sequence in the CCR5 gene (TALC1). (B) hGT1-TALC1 encodes a double-DBD protein including TALC1 linked to Gal4. Tethering of the plasmid to CCR5 is mediated by Gal4 binding to UAS sites found on the plasmid backbone and TALC1 binding to the genomic recognition sequence. Additionally, hGT1-TALC1 encodes a Gal4-PB fusion to draw PB to the plasmid. (C) hGT2-TALC1 encodes a three-part protein consisting of Gal4 linked to TALC1 followed by the PB transposase. Tethering of the plasmid to the CCR5 genomic sequence is made possible by the TALE and Gal4 segment of the protein through binding of Gal4 to UAS sites found on the plasmid backbone. In addition, PB can be relocated to CCR5 via direct linkage to TALC1. (D) hGT3-TALC1 is similar to hGT2-TALC1 except for the TALE and Gal4 DBDs are reversed. Similar to hGT2-TALC1, the TALE and Gal4 segment of the three-part protein mediates the relocation of the plasmid to CCR5. PB is directly linked to the duel DBDs and can therefore also be relocated to the site of interest. (E) hGR1T1-TALC1 encodes a double-DBD including TALC1 linked to a second TALE (TALR1) made to bind specific recognition sites introduced into the plasmid backbone. The double-DBD can therefore simultaneously bind the plasmid and CCR5. hGR1T1-TALC1 also encodes PB linked to TALR1 for the relocation of the transposase to the plasmid backbone and consequently to CCR5. (F) hGT1-TALC1 was modified by replacing TALC1 with a TALE made to bind upstream of TALC1 in the CCR5 gene (TALC2) to make hGT1-TALC2. By combining hG3-TALC1 and hGT1-TALC2 plasmids in a single transfection, both plasmid DNA retargeting and transposase retargeting strategies were used simultaneously to enhance transposition near CCR5. (G) The TALE-localized PB is expected to excise the transposon containing the reporter transgene GFP IRES neomycin (GIN) from the targeting plasmid and integrate nearby. Red arrows indicate PCR primers used to assay for targeted insertion. The depicted genomic primer CCR5 Rev is located 761 bp from the TALC1 recognition site.

Figure 3.

Verification of transposase and TALE activity. (A) Comparison of integration efficiencies between PB constructs transfected into HEK293 cells. One thousand cells were plated and cultured for 3 weeks before G418 resistant/GFP+ colonies were counted. Data are shown as mean values with SD (n = 3). (B) Binding activity of TALE proteins was determined using a transcription factor reporter activation assay in HEK293 cells. TALC1, TALC2 and TALR1 activators were each assayed on luciferase reporter plasmids Rep C1, Rep C2 and Rep R1, which carried a single target site for each TALE activator (n = 4).

Figure 4.

(A) Chromatogram and sequence of PCR product recovered from a representative hG3-TALC1 transfection showing the PB TRE on the left in bold, TTAA junction and flanking genomic CCR5 sequence on the right. (B) Locations of insertion sites recovered in the CCR5 gene. a, hG3-TALC1; b, hGT1-TALC1; c, hGT2-TALC1 (no insertions); d, hGT3-TALC1; e, hGR1T1-TALC1; f, hG3-TALC1 + hGT1-TALC2; g, hG3 (no insertions).

Figure 5.

(A) Cells from a hG3-TALC1 transfection were plated into a 96-well plate, and 1 week later, individual non-overlapping colonies were established for counting; 40× magnification. (B) Nested PCR for the identification of transposition near CCR5. Expected products arose from five positive clones identified from hG3-TALC1 transfection, but not clones from hG3 control transfection. The asterisk denotes a sequenced non-specific PCR product. (C) Transposon copy number for clones 293-1 and 293-2. Quantitative PCR predictions were calibrated using a reference HEK293 cell line known to contain a single copy transposon. (D) Cells positive for GFP reporter gene targeting to CCR5 displayed sustained expression past 10 weeks of culture. Flow cytometry analysis displaying GFP positive events for both untransfected HEK293 cells and an expansion of clone 293–1.