Results: 5

1.
Figure 4.

Figure 4. From: megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering.

megaTAL addressing selectively increases on-target cleavage. (a) I-AniI human genomic near-native sequences and their location in the genome. Synthetic TAL effectors were built against the +9T and +5A+8T sequences underlined in green and the I-AniI near-natives sequences are underlined in blue. (b) Level of gene targeting in TLR cells with endogenous human I-AniI near-native targets using ‘addressed’ +9T and +5A+8T megaTALs and ‘unaddressed’ I-AniI mns. mutNHEJ was not measured via TLR due to the presence of a stop codon in some target sites that prevented flow cytometry readout of these events. (c) Mutation rates at endogenous targets using ‘addressed’ I-AniI megaTALs and ‘unadressed’ mns. The level of mutNHEJ at the endogenous I-AniI near-native targets in 293T cells after 72 h of treatment with mn and megaTAL variants was determined based on MiSeq sequencing results.

Sandrine Boissel, et al. Nucleic Acids Res. 2014 February;42(4):2591-2601.
2.
Figure 2.

Figure 2. From: megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering.

RVD array requirement for megaTAL fusions. (a) Level of cleavage activity (assayed by mutNHEJ, shown in red, and gene targeting, shown in green) of megaTALs built with TAL effectors with different number of repeat units. Cleavage activity of TALE-Zn4-WT megaTALs built with a varying number of RVD array units was normalized to the activity of the WT I-AniI mn (represented by a dashed line) across all cell lines tested. (b) Level of cleavage activity (assayed by mutNHEJ) of megaTALs built with either a codon diverged or nondiverged 6.5 RVD TAL effector array delivered to reporter cells by DNA transfection or lentiviral delivery. Both the codon diverged (CD) and nondiverged (GG) megaTALs were able to rescue the activity of the WT mn using plasmid DNA (pDNA) transfection, lentiviral (LV) or integration-deficient lentiviral (IDLV) transduction. However, delivery using lentiviral vectors resulted in significantly lower activity in cells treated with the nondiverged version of the megaTAL. (c) Proportion of intact (full length) and recombined (short) megaTALs integrated into the genome of 293T cells. Either the codon diverged (CD) or nondiverged (GG) 6.5 RVD WT megaTAL were delivered to cells by lentiviral transduction and the viral integrants were assessed using clonal PCR amplification.

Sandrine Boissel, et al. Nucleic Acids Res. 2014 February;42(4):2591-2601.
3.
Figure 3.

Figure 3. From: megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering.

megaTAL addressing acts by increasing the effective mn concentration at the addressed target site. (a) Level of cleavage activity (assayed by mutNHEJ) of megaTALs built with mns with varying affinities in TLR cell lines containing targets with different DNA spacer lengths. The level of mutNHEJ for each megaTAL tested was normalized to the activity of Y2 (represented by green line) in the same TLR cell line. The average activity of the WT and F13Y mns across each cell line are represented by blue and orange lines, respectively. (b) Effect of megaTAL addressing on activity of Y2 I-AniI toward DNA targets for which it exhibits different biochemical properties. Left panel: Wild-type and singly substituted I-AniI targets and previously determined kcat* and KM* values. Right panel: Mutagenic NHEJ and gene targeting of Y2 and Y2 megaTAL at each target assayed by TLR. Representative flow plots for data from (b) are shown for the Y2 mn (left column) and megaTAL (right column) against high KM* (c) and low kcat* (d) targets. Numbers inside the gates give the percent of mCherry and GFP positive cells and the inset graphs show BFP positive cells on which the experiment was gated.

Sandrine Boissel, et al. Nucleic Acids Res. 2014 February;42(4):2591-2601.
4.
Figure 1.

Figure 1. From: megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering.

DNA spacer requirement for megaTAL fusions. (a) Schematic representation of a megaTAL. The megaTAL architecture involves fusion of a TAL effector with truncated N- and C-terminal domains through a short linker to the N-terminus of a mn (I-AniI, which displays a KD of approximately 90 nM for its cognate target site). Aligned below the megaTAL schematic is the DNA sequence for the L538-I-AniI megaTAL target with a spacer length of 7 bp, the L538 DNA target underlined in green, the I-AniI DNA target underlined in blue and the DNA spacer separating the two outlined with a black box. (b) Increase in levels of mutagenic NHEJ (mutNHEJ) and gene targeting (GT) achieved by I-AniI megaTAL fusion. Results shown are derived from assays of cleavage activity using TLR reporter 293T cells treated 72 h with the L538-Zn4-WT megaTAL (mT) or WT I-AniI mn across targets with different spacer lengths or a ‘scrambled’ (scr) I-AniI target. Cleavage activity for L538-WT megaTAL for a given spacer length is normalized to the activity of the WT mn level (represented by a dashed line) across all cell lines. For all graphs, error bars indicate s.e.m and P-value comparing megaTALs with their appropriate mn counterpart is indicated by asterisk (*P < 0.05, **P < 0.005, ***P < 0.0005).

Sandrine Boissel, et al. Nucleic Acids Res. 2014 February;42(4):2591-2601.
5.
Figure 5.

Figure 5. From: megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering.

megaTAL targeting TCRα achieves extremely high gene disruption with no detectable off-target cleavage in human primary T-cells. (a) Schematic of TCRα megaTAL formed by fusing a synthetic TAL effector built toward the TCRα sequence underlined in green to a designed TCRα mn (target underlined in blue). (b) TCR knockout achieved by parental TCRα mn and TCRα megaTAL in human primary T-cells. Human primary T-cells were transfected with 10 micrograms of RNA encoding the indicated constructs, with or without 10 micrograms of mRNA encoding the Trex2 exonuclease. TCRα gene disruption was assessed by measuring the percentage of cells that transition to being CD3- at 5 days following transfection. Mutation rates at the endogenous TCRα gene were also assessed by Miseq sequencing of amplicons around the cleavage site, and showed indel rates >50%, suggesting that there was a preference for cleavage and disruption of the expressed TCRα allele, particularly when the megaTAL is co-expressed with Trex2. (c) Representative flow cytometry data from transfection of human primary T-cells with TCRα targeting mn or megaTALs +/− Trex2. (d) Heat map showing mutation rates at the endogenous TCRα and putative off-target loci in CD3- primary T-cells treated with the TCRα mn or megaTAL +/− Trex2. Map values were calculated for each locus by subtracting the background rate of indel formation in the control population from that obtained for each nuclease treatment by Miseq sequencing, with the heat map color scale indicated by the key (bottom). Sequencing results across all samples are shown (top), as well as an enlarged view with indel rates at putative off-target (OT) sites in cells treated with the TCRα megaTAL alone (middle). Values calculated as ≤0 are given as ‘0’. Sequencing results were not obtained for putative off-targets OT12 and OT19 and are therefore not shown on the map; other samples for which sequencing results were not obtained are indicated as ‘NA’.

Sandrine Boissel, et al. Nucleic Acids Res. 2014 February;42(4):2591-2601.

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