Transcription Activator-Like Effector Nucleases (TALEN)-Mediated Targeted DNA Insertion in Potato Plants

Abstract

Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. Specifically integrated transgenes are guaranteed to co-segregate, and expression level is more predictable, which makes downstream characterization and line selection more manageable. Because the site of DNA integration is known, the steps to deregulation of transgenic crops may be simplified. Here we describe a method that combines transcription activator-like effector nuclease (TALEN)-mediated induction of double strand breaks (DSBs) and non-autonomous marker selection to insert a transgene into a pre-selected, transcriptionally active region in the potato genome. In our experiment, TALEN was designed to create a DSB in the genome sequence following an endogenous constitutive promoter. A cytokinin vector was utilized for TALENs expression and prevention of stable integration of the nucleases. The donor vector contained a gene of interest cassette and a promoter-less plant-derived herbicide resistant gene positioned near the T-DNA left border which was used to select desired transgenic events. Our results indicated that TALEN induced T-DNA integration occurred with high frequency and resulting events have consistent expression of the gene of interest. Interestingly, it was found that, in most lines integration took place through one sided homology directed repair despite the minimal homologous sequence at the right border. An efficient transient assay for TALEN activity verification is also described.

Keywords: TALEN; double stand break; gene targeting; homologous recombination; potato; transgenic.

FIGURE 1

Schematic of strategy for the targeted T-DNA insertion. The TALEN vector contains a pair of TALENs targeting the endogenous potato Ubi7 intron immediately 5′ to the initiation codon for the first ubiquitin monomer. The LB was removed and an ipt overexpression cassette was put next to the TALEN expression cassettes on the left side of T-DNA. The donor vector contains a promoter-less mutated potato ALS gene (mStALS) next to the RB and the nptII cassette. Upon Agrobacterium-mediated transformation, expression of TALENs generate DSB at the endogenous target, once the donor T-DNA is inserted into this endogenous site, the endogenous promoter will drive the expression of mStALS expression and resulted targeted events can be selected out on herbicide containing medium. The targeted events can also be PCR confirmed by using a primer in the mStALS gene (opened arrow) paired with a primer within the endogenous Ubi7 promoter (solid arrow).

FIGURE 2

Schematic of binary vector pSIM2170 that contains forward and reverse TALENs. Two TALENs were driven by either 35s or FMV constitutive promoter followed by either OCS or Nos terminator, respectively. Each TALEN has a SV40 nuclear localization sequence (NLS), designed repeat variable diresidue (RVD) (E3 or E4 repeat), and truncated C-terminal fused to a FokI cleavage domain. A potato Ubi3 promoter-driven ipt gene cassette was put on the left side of T-DNA and the T-DNA LB was removed in this vector.

FIGURE 3

Verification of designed TALENs in N. benthamiana infiltration. (Top panel) Target vector pSIM2167 of TALENs for N. benthamiana infiltration. A 60 bp DNA sequence from StUbi7 5′ intron was inserted between the first and second amino acid codons of the GUS gene. There is an in frame stop codon (dark gray highlighted) in the spacer region between the two recognition sites (light gray highlighted) which will abolish the GUS protein translation. TALEN-mediated DNA cleavage and subsequent NHEJ based repairing will destroy the stop codon and translation of the GUS will be in frame and restore GUS activity. An AluI restriction enzyme site (underlined) was also included in the spacer to facilitate detection of TALEN-mediated indels. (Middle and bottom panels): The functionality of designed TALEN in transient assay. Target construct was Agro-infiltrated into N. benthamiana leaves alone or together with the TALEN construct. Forty-eight hours after infiltration, leaf tissue at the infiltrated site was collected and stained with GUS staining solution and de-stained with ethanol for microscopic examination. DNA was isolated from co-infiltrated tissue, digested with AluI, PCR amplified, cloned and sequenced. (Middle left panel): Target construct pSIM2167 alone. (Middle right panel): Target construct pSIM2167 co-infiltrated with TALEN construct pSIM2170. (Bottom panel): Sequences of target region with various modifications. The number in the parenthesis represents how many times the modification occurred in the 50 clones sequenced. The numbers on the left are identifiers of the respective clones.

FIGURE 4

Design of knock-in donor vector. The donor vector pSIM2370 T-DNA contains a StUbi7 promoter driven nptII gene cassette at the LB side, and a promoter-less mStALS gene at the RB side. At the sequence level, next to the RB, are sequences of a partial Ubi7 5′ intron region (light gray highlighted), Ubi7 monomer (dark gray highlighted) fused to the mStALS coding sequence (only partial sequence is shown in this figure). The intron sequence contains a spacer between the two TALEN binding sites and the second TALEN recognition site (underlined). There is no ipt expression cassette on the backbone of donor vector.

FIGURE 5

Identification of targeted integration events. DNA from herbicide resistant transgenic lines were isolated for PCR and Southern analyses. (Top panel) Schematic of the Ubi7 locus after site-specific gene integration. Primers 1: within the endogenous Ubi7 promoter and 2: within the mStALS gene coding region are indicated by arrows. Two HindIII restriction enzyme sites, one in the endogenous Ubi7 promoter and one in the mStALS coding region are also labeled. (Middle panel): PCR using primers 1 and 2 detects site-specific integration events. (Bottom panel): Southern blot using StALS probe detects the site-specific integration band at predicted size (boxed). The size of dig-labeled ladders were marked as kb (M: Dig-labeled marker 7, M2: Dig-labeled marker 2).

FIGURE 6

Sequence of LB side flanking region. A pair of primers, one within the endogenous Ubi7 intron and the other within the T-DNA region of the donor vector close to the left border (as shown in Figure 5), were used to amplify targeted insertion in the LB junctions. Amplified products were cloned and sequenced. Sequences of five lines are shown here and aligned to deduce the LB junction after targeted integration. The LB sequence is green highlighted in the deduced sequence with the last three nucleotides red-highlighted, which are lost in the perfect LB cleavage events.