2Department of Clinical Medicine, Department of Cardiological Medicine B, Aarhus University, Aarhus, Denmark
The recombinant adeno-associated virus (rAAV) has proven to be an efficient and attractive tool for targeted genome engineering. Here we present a novel method employing the Golden Gate cloning strategy for fast and efficient construction of rAAV-based gene knockout or single-nucleotide knockin vectors. Two vectors, pGolden-Neo and pGolden-Hyg, were generated as common assembling modules to confer antibiotic resistance to the targeting vector. To validate the method, we then generated two rAAV-based targeting vectors: pAAV-pTP53-KO and pAAV-hTauP301L-KI. Furthermore, we generated a pGolden-AAV plasmid that allows one-step generation of an rAAV-based targeting vector. Our new methodology for rAAV targeting vector assembly is efficient, accurate, time-saving, and cost-effective.
Recombinant adeno-associated virus (rAAV) vectors can efficiently mediate gene disruption in human or porcine cells (1-3). Using rAAV, we had achieved a very high efficiency of targeted knockouts (~35%) of the BRCA1 gene in primary porcine fibroblasts (3, 4). For the BRCA1 targeting vector, we used a simplified method, three-way-fusion PCR, to combine the antibiotic resistance fragment of the pNeDaKO vector with two PCR amplicons comprising the left and right homology arms (1). Though usually successful, we sometimes encountered problems with PCR-induced mutations or difficulties with amplifying the fusion product when using the three-way-fusion PCR method.
We sought an alternative approach that could overcome the limits of three-way-fusion PCR without increasing the complexity of the technique or the time required for constructing the targeting vector. Golden Gate cloning (GGC), which employs type IIS restriction enzymes (e.g., BsaI) and allows user-defined ligation of multiple fragments in one reaction, has been used for the generation of complex vectors such as those used in the transcription activator-like effector nuclease (TALEN) system (5-7). This suggests that the GGC approach might be translated to the generation of rAAV-based gene targeting vectors.
In this study, we generated two common assembly vectors (pGolden-Neo and pGolden-Hyg) that are compatible with GGC and contain a gene conferring resistance to either neomycin (Neo) or hygromycin (Hyg), respectively. We also showed that this method can be used to generate rAAV-based gene knockout or single-nucleotide knockin vectors to be used for genetic engineering. We then produced a pGolden-AAV vector, which enabled us to generate an rAAV-mediated gene targeting vector in one step.
We selected the type IIS restriction enzyme BsaI, which recognizes an asymmetric DNA sequence (5′-GTCTC) and cleaves 1 and 5 bp away from the recognition site in the sense and anti-sense strands respectively, creating a 5′ 4-nucleotide overhang (Figure 1A) (8). First, we generated the assembly vectors, pGolden- Neo and pGolden-Hyg (Figure 1, A and B), for antibiotic-based selection and screening of rAAV-mediated gene targeted cell clones. These two vectors were constructed by amplifying the selection cassettes from the pNeDaKO-Neo and pNeDaKO-Hyg vectors (1), which confer resistance to neomycin (Neo) or hygromycin (Hyg), respectively. However, we excluded the gene conferring zeomycin resistance (Zeo) in the pNeDaKO-Neo and pNeDaKO-Hyg vectors from our pGolden-Neo and pGolden-Hyg vectors, as the vector cloning efficiency was sufficiently high even without Zeo-based selection. By excluding the Zeo selection cassette, we could increase the length of the homology arms by ~450 bp without compromising the limited genomic cargo size of rAAV (max 4.7 kb). As with the pGolden-Neo and pGolden-Hyg vectors, the left homology arm (LHA) and right homology arm (RHA) can be generated by PCR with each primer comprising a BsaI site and a unique linker added to the 5′ end (Figure 1C). The PCR product was sub-cloned into a Topo vector by blunt-end cloning. The construct was produced in E.coli and then sequenced to validate its integrity. In principle, the gene targeting cassette can be generated in one step as illustrated in Figure 1C. The advantage of using the GGC strategy is that BsaI digestion and T4 ligase-guided ligation can be carried out in one reaction. A detailed protocol for the method is provided in the Supplementary Material.
In this study, we developed a time-saving and cost-effective method for constructing rAAV-mediated gene targeting vectors in a single assembling reaction using our modular vectors: pGolden-Neo (Addgene ID 51422), pGolden-Hyg (Addgene ID 51423), and pGolden-AAV (Addgene ID 51424).
The GGC method for rAAV targeting vector construction was applied in the generation of an rAAV-based porcine TP53 knockout vector. TP53 is a tumor suppressor gene that plays a crucial role in carcinogenic processes (9). Porcine models with TP53 mutations (e.g., knockouts or TP53R167H) have been created to study the tumorigenic role of TP53 in cell transformation and chemoresistance (10). We have generated porcine models for studying human cancers, such as the BRCA1 knockout pigs (3, 4). It is very likely that homozygosity for BRCA1 null mutations will be embryonic lethal in pigs as they are in rodents. In mice, embryonic lethality can be rescued by TP53 inactivation (11). Thus, in order to generate viable BRCA1 homozygous knockout pigs, we designed an rAAV targeting vector that deleted exons 5–7 and part of exon 8 of the porcine TP53 gene upon homologous recombination (Figure 2A). In this study, we only demonstrated the application of the pGolden- Neo assembly vector for GGC. However, the method described here can be directly applied to the pGolden-Hyg assembly vector.
The first step of constructing an rAAV targeting vector with the GGC method required, in addition to careful primer design, bioinformatic analyses to avoid including repetitive DNA sequences or restriction enzyme (NotI and BsaI) recognition sites in the homology region (see detailed protocol in the Supplementary Material). Then, the LHA and RHA were amplified from the porcine TP53 (pTP53) gene (Figure 2B). The resulting PCR products were gel-purified and sub-cloned into a Topo vector with the Zero Blunt TOPO PCR Cloning Kit (Life Technologies, Naerum, Denmark) (Figure 2C), followed by sequencing of purified plasmid DNA to confirm that mutations had not been introduced in the homology arms by the PCR amplifications. Alternatively, the gel-purified LHA and RHA PCR products can also be used for GGC assembling without Topo cloning. However, this will require sequencing of the entire pFUS-pTP53-KO plasmid, which is not necessary when using the Topo cloning strategy. As shown in Figure 2D, the pFUS-pTP53-KO vector comprising the homology arms and selection cassette could be visualized by 1% agarose gel electrophoresis after GGC. The upper band in the gel (Figure 2D) represents duplex concatemer ligation. The efficiency of the procedure was almost 100%, as demonstrated by transformation of the GGC product into competent cells (Figure 2E) and subsequent PCR screening of transformed bacterial clones (Figure 2F). The pFUS-pTP53-KO plasmid was further validated by NotI restriction enzyme digestion (Figure 2G) and sequencing (data not shown). The final pAAV-pTP53-KO targeting vector was constructed by digesting the pFUS-pTP53-KO vector with NotI and ligating the TP53 targeting fragment into the pAAV-MCS NotI fragment (data not shown).
We then explored the possibility of using the GGC method to generate an rAAV-basedknockin vector for targeting single nucleotides. The microtubule-associated protein tau (MAPT) gene is associated with several neurodegenerative disorders, including Alzheimer's disease. Multiple mutations (including TauP301L) were identified within the MAPT gene in a large number of families with hereditary frontotemporal dementia and parkinsonism linked to chromosome 17, as reviewed by Lee et al. (12). Overexpression of the TauP301L mutation in mice causes motor impairment and cognitive decline (13, 14). The P301 residue of human tau is encoded in exon 10 of the MAPT gene (Figure 3A). We designed the rAAV-based TauP301L targeting vector by changing the nucleotide sequences in the LHA from CCG to CtG (Figure 3A). Instead of using a single-nucleotide mutagenesis kit, we constructed the LHA with two continuous PCR fragments: LHA1 and LHA2. The CtG mutation was introduced automatically using the predefined linker CtGG, as highlighted in yellow (Figure 3B). Similar to the protocol described above for constructing the TP53 knockout vector, we amplified the LHA1, LHA2, and RHA from the human MAPT gene (Figure 3C), sub-cloned the PCR products into Topo vectors, and sequenced the vectors (data not shown). Then the pTopo-LHA1, pTopo-LHA2, pTopo- RHA, and pGolden-Neo vectors were assembled into the pFUS-A vector using the GGC procedure (Figure 3D) followed by PCR screening of the resulting transformed white bacterial clones with screening primers (Figure 3E and Supplementary Table S1). The pFUS-hTAUP301L-KI vector was further validated by restriction enzyme digestion with NotI and subsequent sequencing (Figure 3F). The final pAAV-hTAUP301L-KI vector was generated by sub-cloning the hTAUP301L-KI fragment into the pAAV-MCS NotI fragment.
To further simplify the cloning procedure, we modified the pAAV-MCS plasmid to skip the second sub-cloning step. There is one BsaI recognition site in the ampicillin resistant coding region in the pAAV-MCS plasmid, which hampers the use of pAAV-MCS for BsaI-based GGR. We used a fusion PCR-based mutagenesis strategy (primers given in Supplementary Table S1) and generated a modified pAAV-MCS plasmid, named pGolden-AAV plasmid (Figure 4A), which can replace the pFUS-A backbone vector used in the first GGR (Figure 1C). Thus, generation of the rAAV-mediated targeting vector was accomplished in one GGR step (Figure 4B). As anexample, we used this method to construct the pAAV-pTP53-KO targeting vector (Figure 4C). Methods used for rAAV virus packaging, viral transduction, and screening for gene-targeted cloned cells have been described previously (2, 3, 15). The pGolden-Neo, pGolden-Hyg, and pGolden-AAV plasmids have been deposited in Addgene.
In conclusion, we have developed a GGC method for efficient construction of rAAV-based gene knockout or single-nucleotide knockin vectors. Furthermore, this method can also be applied to generate plasmid-based donor vectors for TALEN- or CRISPR/ Cas9-mediated gene targeting by homologous recombination. Using this method, an rAAV-based targeting vector can be constructed in three working days. Author contributions
Y.L. conceived the idea, developed the protocol, and performed experiments. L.L. participated in the design and generation of the pGolden-AAV plasmid. L.B. and C.B.S. provided support to the project. Y.L. wrote the manuscript. L.B. and C.B.S. revised the manuscript.
We are grateful to Trine Skov Petersen for technical assistance, as well as Bert Vogelstein and Kenneth W. Kinzler at John Hopkins University for kindly providing the pNeDaKO plasmid vectors. This work was supported by the Fogh-Nielsen Prize (to Y.L.), the Danish National Advanced Technology Foundation, and the annuum grant from Aarhus University (to L.B.).
The authors declare no competing interests.
Address correspondence to Yonglun Luo, Department of Biomedicine, Faculty of Health, Aarhus University, Aarhus, Denmark. E-mail: [email protected]
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