Single-strand nicks induce homologous recombination with less toxicity than double-strand breaks using an AAV vector template

Abstract

Gene targeting by homologous recombination (HR) can be induced by double-strand breaks (DSBs), however these breaks can be toxic and potentially mutagenic. We investigated the I-AniI homing endonuclease engineered to produce only nicks, and found that nicks induce HR with both plasmid and adeno-associated virus (AAV) vector templates. The rates of nick-induced HR were lower than with DSBs (24-fold lower for plasmid transfection and 4- to 6-fold lower for AAV vector infection), but they still represented a significant increase over background (240- and 30-fold, respectively). We observed severe toxicity with the I-AniI 'cleavase', but no evidence of toxicity with the I-AniI 'nickase.' Additionally, the frequency of nickase-induced mutations at the I-AniI site was at least 150-fold lower than that induced by the cleavase. These results, and the observation that the surrounding sequence context of a target site affects nick-induced HR but not DSB-induced HR, strongly argue that nicks induce HR through a different mechanism than DSBs, allowing for gene correction without the toxicity and mutagenic activity of DSBs.

Figure 1.

Nicks induce HR with a transfected template. (A) Schematic of integrated target site CnZPNOA3, with a lacZ gene inactivated by the introduction of the I-AniI recognition site (black box). The repair template plasmid (pA2nZ3113) is shown above with regions of homology indicated by a dotted ‘X’. (B) β-gal⁺ cells per cm² in confluent polyclonal 293/CnZPNOA3 cells transfected with one of the plasmids expressing either GFP or the inactive, cleavase or nickase I-AniI and template plasmid pA2nZ3113 (except for the GFP−template condition, in which cells were transfected with only GFP-expressing plasmid). Values are means of four experiments ± SE.

Figure 2.

Expression of I-AniI cleavase generates DSBs, but nickase does not. (A) Schematic of the integrated target site CnZPNOA3, containing the I-AniI recognition site, with probe and enzyme cleavage sites marked. (B) Genomic DNA was extracted from 293/CnZPNOA3 cells 3 days after transduction with I-AniI-expressing lentivirus—either inactive, cleavase or nickase. DNA was digested in vitro with either SpeI alone or SpeI and purified I-AniI cleavase before being used for the Southern blot. Size markers were generated by in vitro digestion of pCnZPNOA2 plasmid with SpeI and SpeI/I-AniI and gel extraction of 4.0- and 1.5-kb bands, respectively. (C) Cell lysate was collected from 293/CnZPNOA3 cells 3 days after transduction with the same I-AniI-expressing lentivirus preparations used for the Southern blot and further HR assays. An amount of 10 µg of each lysate was loaded for western analysis and anti-HA antibody (mouse mAb 6E2, Cell Signaling Technology) was used to detect the HA-tagged I-AniI proteins.

Figure 3.

Analysis of potential NHEJ mutations using a sensitive qPCR-based assay. (A) Schematic of the integrated target site CnZPNOA3 showing the positions of the primers used for qPCR: lacZqF1 (F1), lacZqR1 (R1), lacZqF2 (F2) and lacZqR2 (R2). (B) Table showing the ratio of qPCR amplification with primer pair 2 to primer pair 1 after digest of genomic DNA with I-AniI, analysis of the fraction of the qPCR products which cannot be cleaved by I-AniI, and the final estimated percentage of mutated I-AniI sites (a product of the first two columns). Values are means of two experiments calculated independently (individual values for nickase-induced mutation were 120- and 180-fold less than that with cleavase). (C) Primer pair 2 qPCR products were digested with I-AniI, and a representative agarose gel revealing the digested and undigested products is shown.

Figure 4.

Nicks induce HR with an AAV vector template with less toxicity than DSBs. (A) Schematic of integrated target site CnZPNOA3, with a lacZ gene inactivated by the introduction of the I-AniI recognition site. The AAV2-nZ3113 genome used as an HR template is shown above with homology indicated by a dotted ‘X’. (B) β-gal⁺ foci counted 9 days after infection of 5 × 10⁴ cells with 1.5 × 10⁴ vg/cell AAV2-nZ3113. Cells were transduced with different MOIs of the three I-AniI-expressing lentivirus vectors 3 days prior to AAV vector delivery. (C) Survival of cells transduced with different MOIs of the three I-AniI-expressing lentivirus vectors determined by counting Coomassie-stained colonies 9 days after AAV vector infection and normalized to colony-forming ability of untransduced cells assayed in parallel. Survival points and β-gal⁺ bars represent means of three experiments ± SE, each conducted with independent polyclonal cell populations. White bars and white squares represent inactive I-AniI-expressing lentivirus; black bars and black diamonds, I-AniI cleavase; gray bars and gray circles, I-AniI nickase. (D) β-gal⁺ foci counted 9 days after infection of 5 × 10⁴ cells with varying amounts of AAV2-nZ3113. All cells were transduced with I-AniI-expressing lentivirus vectors 3 days earlier at an MOI of 1 TU/cell. White bars represent mock-lentiviral infection; black bars, cleavase; gray bars, nickase.

Figure 5.

Timing of endonuclease delivery. β-gal⁺ foci counted 9 days after infection of 293/CnZPNOA3 cells with 2 × 10⁴ vg/cell of AAV2-nZ3113. In all cases, cells were seeded at 5 × 10⁴ cells per well in 24-well plates the day before AAV vector infection. Cells were transduced with one of three I-AniI-expressing lentivirus vectors (at an MOI of 2.5 TU/cell) at three different times: 3 days before, the same time as, or 3 days after AAV vector infection. Results are means of two experiments, except for the transduction 3 days after AAV vector infection and the nickase transduction at the same time as AAV vector infection, which are means of three experiments. White bars represent inactive I-AniI-expressing lentivirus; black bars, I-AniI cleavase; gray bars, I-AniI nickase.

Figure 6.

Target design affects nick-induced HR, but not DSB-induced HR. (A) Schematic of the differences between the insertion (CnZPNOA2) and replacement (CnZPNOA3) targets with homology to wt lacZ. The I-AniI recognition site is capitalized and in bold for both targets. (B) β-gal⁺ cells/cm² after transfection as in Figure 2, using 293 cells with integrated insertion target (CnZPNOA2, white bars) or replacement target (CnZPNOA3, black bars). Means of four experiments ± SE. (C) β-gal⁺ foci after transduction with cleavase-expressing lentivirus and infection with AAV2-nZ3113 as in Figure 3. Means of three experiments ± SE. (D) β-gal⁺ foci after transduction with nickase-expressing lentivirus and infection with AAV2-nZ3113 as in Figure 3. Means of three experiments ± SE.

Figure 7.

In vitro comparison of replacement and insertion targets. (A) Frequency of linearized plasmid containing either the insertion target (pnZA2, white diamonds) or replacement (pnZA3, black squares) following in vitro digestion with 1 µM purified I-AniI cleavase at 37°C for 0–2 h. (B) Frequency of nicked plasmids with 1 µM purified I-AniI nickase. (C) Frequency of linearized plasmid with both targets with increasing concentrations of purified I-AniI cleavase for 2 h at 37°C in vitro. (D) Frequency of nicked plasmid with increasing concentrations of purified I-AniI nickase for 2 h at 37°C in vitro.

Figure 8.

Model for HR at DSBs and nicks. When either the replacement or insertion target sites are cleaved, the ends will be processed and the AAV vector template will invade and align with one of the broken strands. When the replacement and insertion sites are nicked, however, the alignment of the target and AAV vector template is different. The nicked target can align with a single-stranded AAV vector template of either ‘+’ or ‘–’ orientation, followed by Holiday junction resolution and completion of HR. Notably, in nick-induced HR with either template there is a step in which an intact heterodimer is present (marked with a gray circle). This does not occur if both strands are cut. We hypothesize that the discordant activity observed in Figure 6 could be explained by discrimination at the level of resolution of this heterodimer.