Genome targeting by hybrid Flp-TAL recombinases

Abstract

Genome engineering is a rapidly evolving field that benefits from the availability of different tools that can be used to perform genome manipulation tasks. We describe here the development of the Flp-TAL recombinases that can target genomic FRT-like sequences in their native chromosomal locations. Flp-TAL recombinases are hybrid enzymes that are composed of two functional modules: a variant of site-specific tyrosine recombinase Flp, which can have either narrow or broad target specificity, and the DNA-binding domain of the transcription activator-like effector, TAL. In Flp-TAL, the TAL module is responsible for delivering and stabilizing the Flp module onto the desired genomic FRT-like sequence where the Flp module mediates recombination. We demonstrate the functionality of the Flp-TAL recombinases by performing integration and deletion experiments in human HEK-293 cells. In the integration experiments we targeted a vector to three genomic FRT-like sequences located in the β-globin locus. In the deletion experiments we excised ~ 15 kilobases of DNA that contained a fragment of the integrated vector sequence and the neighboring genome sequence. On average, the efficiency of the integration and deletion reactions was about 0.1% and 20%, respectively.

Figure 1

Flp-TAL recombinases. (A) General mode of target binding by Flp-TAL recombinases. (B) Flp-TAL and TAL-Flp arrangements of the Flp and TAL modules and the respective possible relative arrangements of the Flp and TAL recognition sequences. (C) Schematic of the Flp-TAL recombinase.

Figure 2

FRT-like sequences FL-61, FL-63, and FL-71. (A) Relative location of the FL-61, FL-63, and FL-71 sequences in the human genome. (B) Alignment of FRT, FL-61, FL-63, and FL-71. Base pairs in FL-61, FL-63, and FL-71 that differ from the corresponding base pairs in FRT are shown as lower case bold black letters. (C) FL-61_TAL, FL-63_TAL, and FL-71_TAL sequences. 5′T bases for the TAL recognition sequences (marked by blue arrows) are shown in red.

Figure 3

Evolution of Flp-TAL recombinases. (A) The inversion assay. The reporter bears the inversion cassette flanked by the recombination targets in the head-to-head orientation: FL-61, FL-63, or FL-71 (marked as RT) and FRT*, which has the spacer either from FL-61, FL-63, or FL-71, respectively. Upon the expression of a recombination-competent Flp variant, the cassette is inverted so the gene that encodes this variant can be PCR amplified (the small black arrows above the Flp variant gene and the inversion cassette indicate the location and orientation of the PCR primers). (B) The deletion assay. The deletion reporter bears the lacZα cassette flanked by the respective recombination targets in the head-to-tail orientation. If a Flp variant is able to delete the cassette, bacterial cells, in which the deletion event occurs, form white/partially white colonies when plated on the X-gal containing plates. (C) Mutations in the FV61, FV63, and FV71 variants. (D) Activity of FV61, FV63, and FV71 on the FL-61, FL-63, and FL-71 substrates. The assays were performed using the respective deletion reporters. (E) Electrophoregram of the plasmid DNA isolated from the colonies that were pooled from the respective plates shown in (D); the plasmid DNA was digested with HindIII that uniquely cuts the expression and reporter plasmids. Ctr, control vectors and vector combinations: Exp, Flpe expression vector; Rep, the deletion reporter that bears the lacZα cassette flanked by the FRT sequences; Exp + Rep, Flpe expression vector and the respective reporter, in which the lacZα cassette was completely deleted; M, DNA ladder (NEB, 2-log). The arrows below the electrophoregram point to the location of the respective vectors.

Figure 4

Flp-TAL variants target FRT-like sequences FL-61, FL-63, and FL-71 in their native chromosomal environment. (A) Schematics of the integration assays. The reporter pTarget can be integrated either into FL-61, FL-63, or FL-71 depending on the specificity of the Flp-TAL recombinase and the version of pTarget: FL-61/FL-71 or FL-63/FL-71. Upon integration of pTarget into FL-61 or FL-63 the resultant cells become hygromycin resistant and red, while upon integration into FL-71, the cells become hygromycin resistant and green (images of the individual expanded hygro^R/red and hygro^R/green colonies are shown as examples). The analysis of the individual colonies was performed in two biological replicates. LJ and RJ mark the locations of the left and right junctions of the integrated reporter and genomic DNA, respectively; diagnostic PCR at these locations was used to determine the authenticity of the reporter integration. (B) PCR analysis of the pooled hygro^R colonies generated in the experiments with the Flp-TAL recombinase, the ‘plain’ recombinase variant, and the ‘empty’ expression vector. LJ, RJ, the PCR analysis of the left and right junctions of pTarget integrated into the respective genomic sequences; M, DNA ladder (NEB, 2-log). (C) PCR analysis of the pooled hygro^R colonies generated in the integration experiments with three combinations of the FV71-TAL recombinases: FV71-TAL(L24) + FV71-TAL(R15), FV71-TAL(L18) + FV71-TAL(R15) and FV71-TAL(L15) + FV71-TAL(R15). The diagnostic PCR analysis was performed at the right junction.

Figure 5

Target specificity of the Flp variants within Flp-TAL is determined by the TAL module. (A) pTarget co-transfected with either FV61-TAL, FV63-TAL, or FV71-TAL into HEK-293 cells efficiently targets only the intended FRT-like sequence. (B) The TAL modules in the Flp-TAL recombinases FV71-TAL61 and FV71-TAL63 variants deliver the FV71 variant to the FRT-like sequences FL-61 and FL-63 but not to FL-71. The diagnostic PCR analyses were performed at the respective right junctions between the integrated pTarget and genomic DNA. M, DNA ladder (NEB, 2-log).

Figure 6

Flp-TAL recombinase can delete large genome DNA fragments. (A) Schematic of the deletion assay. Upon expression of FV71-TAL in cells, in which pTarget was pre-integrated into FL-61 (Fig. 4A), the DNA fragment located between two FL-71 sequences can be deleted (this DNA fragment contains ~ 4.7 kb of reporter DNA and ~ 10 kb of genome DNA). (B) PCR analysis of the FV71-TAL treated cells. The deletion activity was analyzed in three biological replicates (see Supplemental Fig. 4). FL-61(LJ) and FL-71(RJ), the PCR analysis of the respective left and right vector-genomic junctions of the integrated pTarget before and after the treatment with FV71-TAL. M, DNA ladder (NEB 2-log). (C) Sequencing of the deletion-specific PCR product FL-71(RJ) confirmed its identity.