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. 2011 Nov;39(21):9283-93.
doi: 10.1093/nar/gkr597. Epub 2011 Aug 3.

A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity

Claudio Mussolino  1 , Robert Morbitzer, Fabienne Lütge, Nadine Dannemann, Thomas Lahaye, Toni Cathomen
Affiliations

A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity

Claudio Mussolino et al. Nucleic Acids Res. 2011 Nov.

Abstract

Sequence-specific nucleases represent valuable tools for precision genome engineering. Traditionally, zinc-finger nucleases (ZFNs) and meganucleases have been used to specifically edit complex genomes. Recently, the DNA binding domains of transcription activator-like effectors (TALEs) from the bacterial pathogen Xanthomonas have been harnessed to direct nuclease domains to desired genomic loci. In this study, we tested a panel of truncation variants based on the TALE protein AvrBs4 to identify TALE nucleases (TALENs) with high DNA cleavage activity. The most favorable parameters for efficient DNA cleavage were determined in vitro and in cellular reporter assays. TALENs were designed to disrupt an EGFP marker gene and the human loci CCR5 and IL2RG. Gene editing was achieved in up to 45% of transfected cells. A side-by-side comparison with ZFNs showed similar gene disruption activities by TALENs but significantly reduced nuclease-associated cytotoxicities. Moreover, the CCR5-specific TALEN revealed only minimal off-target activity at the CCR2 locus as compared to the corresponding ZFN, suggesting that the TALEN platform enables the design of nucleases with single-nucleotide specificity. The combination of high nuclease activity with reduced cytotoxicity and the simple design process marks TALENs as a key technology platform for targeted modifications of complex genomes.

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Figures

Figure 1.
Figure 1.
Minimal DNA binding domain of the AvrBs4 TALE. (a) Schematic of AvrBs4 and AvrBs3 deletion variants. The black rectangles represent the 17.5 central tandem repeat arrays that mediate DNA recognition. The C-terminal nuclear localization signals (NLS) and transcriptional activation domain (AD) are highlighted in gray. Deletion variants are generated using the restriction sites indicated with letters (B: BamHI; N: NarI; E: Eco147I; H: HincII; C: BclI) and reported in the respective names (right). The number of remaining residues at N- and C-termini (relative to the position of the DNA binding domain) and the RVDs with the expected target sequences for AvrBs3 and AvrBs4 are shown. (b) Transcriptional reporter assay. Designer TALE-based transcription factors (dTALEs) consist of the VP16 transcriptional AD fused to the AvrBs4 or AvrBs3 deletion variants. HEK293T cells were transfected with dTALE expression plasmids and the reporter construct containing two upstream binding sites for AvrBs4 (AvrBs4), followed by a minimal promoter element and the luciferase gene. The graph displays luciferase activity normalized for transfection efficiency and relative to transfection with empty vector (–). Significant activation above background is indicated by *P < 0.05 and **P < 0.01. (c) dTALE expression levels. Transfected HEK293T cells were harvested after 48 h and cell lysates probed either with antibodies against HA tag or β-actin. Lysate of non-transfected cells is marked with ‘–’.
Figure 2.
Figure 2.
Optimal spacer length for DNA cleavage by TALEN. (a) In vitro cleavage activity. TALENs consist of the TALE repeat units preceded by an HA tag and an NLS. The C-terminal catalytic domain of the FokI endonuclease (FokI) mediates cleavage after dimerization of two TALEN subunits. The linear DNA substrate containing an inverted AvrBs4 repeat was incubated with TALENs in vitro and the extent of cleavage analyzed by agarose gel electrophoresis. The respective spacer lengths (left) and the positions of the cleavage products (arrows) are indicated. (b) Cleavage activity in cellula. HEK293T cells were co-transfected with the respective TALEN expression plasmids and reporter plasmids that harbors an inverted repeat of AvrBs4 integrated into the 5′-end of a gene encoding a destabilized EGFP (dsEGFP). The position of a binding site for I-SceI used for internal reference is shown. The graph displays reduction of EGFP mean fluorescent intensity (MFI) relative to a non-functional nuclease, as determined by flow cytometry. The respective spacer lengths separating the AvrBs4 sites are indicated. (c) Relative TALEN activity in relation to spacer length. The graph displays TALEN activity (from [b]) relative to I-SceI at the various spacers. (d) TALEN expression levels. Transfected HEK293T cells were harvested after 48 h and cell lysates probed either with antibodies against HA tag or β-actin. Lysate of non-transfected cells is marked with ‘–’.
Figure 3.
Figure 3.
Comparison of activity versus toxicity profiles of TALEN and ZFN. (a) Designer nuclease-mediated gene disruption. The HEK293-based reporter cells harbor an integrated dsEGFP gene that contains an inverted heterodimeric AvrBs4/AvrBs3 target sequence separated by 13-bp spacer in the 5′-end of the open reading frame. The position of the diagnostic XhoI site is indicated. For internal reference, a binding site for I-SceI was placed downstream of the TALEN target site. The graph shows the percentage of EGFP-negative cells 5 days after transfection with the nuclease expression vectors (TALEN, ZFN or I-SceI). (b) Molecular characterization. Genomic DNA was extracted 5 days after transfection and PCR amplicons encompassing the target sites were used as templates for digestion with XhoI. An arrow indicates the position of the XhoI-resistant DNA fragment. The numbers below designate the percentages of XhoI-resistant PCR fragments (note background level of ~6%). (c) Activity versus toxicity. The EGFP reporter cells were transfected with increasing amounts (1–600 ng) of nuclease expression vectors (TALEN, ZFN or I-SceI) and a mCherry-encoding plasmid. The percentage of EGFP and mCherry-positive cells was determined by flow cytometry after 2 and 5 days. The graphs display gene disruptions activities (EGFP-negative cells at Day 2; top) and nuclease-associated cytotoxicities (fraction of mCherry-positive cells at Day 5 as compared to Day 2 after transfection; bottom), relative to cells transfected with a mock plasmid. Statistically significant differences in toxicities between TALEN and ZFN are indicated by *P < 0.05 or **P < 0.01, respectively.
Figure 4.
Figure 4.
Modifications at the human IL2RG locus. (a) Target sites in human IL2RG gene. Target sites are highlighted by gray shaded (TALEN) or black (ZFN) boxes, respectively. The RVDs of the engineered TALEs as well as the expected target sequences are indicated. (b) Comparison of the activities and toxicities of IL2RG-specific designer nucleases. HEK293T cells were co-transfected with expression plasmids-encoding IL2RG-specific TALEN and ZFN, respectively, and a reporter plasmid harboring an IL2RG-dsEGFP fusion gene. The graphs display reduction of the mean fluorescent intensity (MFI) of IL2RG-dsEGFP (left) and relative cell survival as compared to cells expressing a nonfunctional nuclease indicated with ‘–’ (right). NH and NC designate the TALEN scaffold used. Statistically significant differences in activity and toxicity between the TALEN and ZFN tested are indicated by *(P < 0.05) or **(P < 0.01), respectively. (c) Disruption of endogenous human IL2RG locus. After transfection with TALEN (NC scaffold) and ZFN expression vectors, genomic DNA was extracted and used as a template for PCR amplification. Amplicons encompassing the target sites were digested with the mismatch-sensitive T7 endonuclease 1 (T7E1). Arrows indicate the expected positions of the T7E1 digestion products. Numbers at the bottom designate the average percentage of modified alleles (n = 2). TALENs targeting the CCR5 locus (C5-TALEN) were used as negative controls. L and R refer to the nuclease subunits, binding to the left or right target half-sites, respectively.
Figure 5.
Figure 5.
Modifications at the human CCR5 locus. (a) Target sites in human CCR5 gene. Target sites are highlighted by gray shaded (TALEN) or black (ZFN) boxes, respectively. The RVDs of the engineered TALEs as well as the expected target sequences are shown. Bold letters designate the nucleotides not conserved in the CCR2 locus. (b) Disruption of endogenous human CCR5 locus. After transfection with TALEN and ZFN expression vectors, genomic DNA was extracted and PCR amplicons encompassing the CCR5 target sites digested with T7E1. Arrows point out the expected positions of the T7E1 digestion products. Numbers at the bottom indicate the average percentage of modified alleles (n = 3). TALENs targeting the IL2RG locus (GC-TALEN) were used as a negative control. NH and NC designate the TALEN scaffold. (c) Off-target activity at CCR2 locus. Asterisks in the alignment of CCR5 and CCR2 designate the mismatches between the two target sequences, bold letters the nucleotides affecting binding of the designer nucleases. PCR amplicons encompassing the CCR2 target locus were digested with T7E1. Arrows point out the expected positions of the T7E1 digestion products. Numbers at the bottom designate the average percentage of modified alleles (n = 2). (d) Cytotoxicity of CCR5-specific designer nucleases. The graph displays nuclease-associated cytotoxicities relative to cells expressing a nonfunctional nuclease (–). Statistically significant differences in toxicities between TALENs and ZFN are indicated by **P < 0.01.
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