The use of CRISPR/Cas9 technology can be limited by delivery options for Cas9 and the single guide RNA (sgRNA). Transfection of cells with plasmids encoding Cas9 and sgRNA is the most commonly used method. However, many human cell types are considered hard-to-transfect, making plasmid-based delivery difficult. One alternate strategy for delivering CRISPR/Cas9 components to these cell types is viral transduction.
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Improved CRISPR/Cas9 genome editing in hard-to-transfect mammalian cells using AAV
- Viruses encoding truncated portions of Cas9 with a 1.6-kb region of homology are prepared in HEK 293T packaging cells:
Recombination in target cells results in a full-length Cas9 gene expression cassette
- An innovative method to deliver the large Cas9 gene with a small virus:
To overcome the size limitation of AAV, the Cas9 gene is divided between two vectors
- AAV-mediated delivery of Cas9 and sgRNA expression cassettes results in more indels, especially in hard-to-transfect cell lines:
A higher rate of mutation is obtained with AAV-mediated delivery of Cas9 and sgRNA compared to transfection-based delivery
Advantages of AAV for CRISPR/Cas9 delivery
Recombinant adeno-associated virus (AAV) has several advantages over other types of viruses for gene delivery. Importantly, AAV does not integrate into the host genome, precluding genomic integration and sustained expression of Cas9, thereby reducing the likelihood of Cas9 off-target effects. AAV also exhibits lower immunogenicity and has a small genome relative to other non-integrating viruses (e.g., adenovirus), making it easier to manipulate.
The small size of the AAV genome can be a limitation when packaging a large gene such as the S. pyogenes Cas9 (SpCas9). The 4.1-kb SpCas9 gene together with its optimal promoter and polyadenylation signal exceeds the capacity that can be efficiently packaged into AAV viral particles (Byrne, Mali, and Church 2014). To overcome this limitation, we developed a system that takes advantage of DNA recombination that is inherent to AAV genome processing (Hirsch, Agbandje-McKenna, and Samulski 2010). The vectors included with the AAVpro CRISPR/Cas9 Helper Free System (AAV2) split the Cas9 gene into two portions (Cas9-Up and Cas9-Down) that have a 1.6-kb shared region of homology (Figure 1). This homologous region mediates recombination with high efficiency inside the target cell, thereby producing a full-length Cas9 expression cassette.
The AAVpro CRISPR/Cas9 Helper Free System (AAV2) is a complete system for the delivery of Cas9 and a gene-specific sgRNA to mammalian cells using AAV2 (AAV serotype 2). First, oligos encoding a gene-specific guide sequence are annealed to form duplexes. The duplexed DNA is ligated to the pre-linearized pAAV-Guide-it-Down plasmid (Figure 2B). All the components needed for the annealing and ligation of the oligos are provided. AAV2-Up and AAV2-Down viruses are packaged independently in a HEK 293T packaging cell line. Packaging cells are transfected with the respective pAAV-Guide-it-Up and pAAV-Guide-it-Down plasmids, and the pHelper and pRC2-mi342 plasmids that encode viral packaging components and miRNA-342, a human microRNA that increases AAV2 titer. AAV2 viral particles are then extracted using the included AAVpro Extraction Solution. Target cells are then transduced with equal number of AAV2-Up and AAV2-Down viruses to ensure efficient recombination. Recombination between Cas9-Up and Cas9-Down within target cells results in a full-length Cas9 gene expression cassette. Following transcription and translation, Cas9 is guided to the target genomic locus by the sgRNA, where it creates a double-stranded break.
Produce consistent and equivalent high-titer AAV2-Up and AAV2-Down virus
To ensure efficient genome editing, obtaining high and equivalent titers of both AAV2-Up and AAV2-Down virus is critical. AAV2-Up and AAV2-Down viral particles encoding truncated portions of Cas9 and a sgRNA targeting CCR5 were generated in three independent experiments using the AAVpro CRISPR/Cas9 Helper Free System (AAV2) (Figure 4). Identically high titers were obtained for both viruses. The AAVpro Extraction Solution, which is provided with the kit, results in viral yields that are 3 times higher than those obtained with traditional freeze-thaw methods.
The CCR5 gene, which encodes a cell surface chemokine receptor, was targeted for CRISPR/Cas9 editing. Oligos containing a guide sequence targeting the CCR5 gene were designed, annealed, and ligated to the pAAV-Guide-it-Down plasmid. AAV2-Up and AAV2-Down viruses were packaged following the recommended protocol. HEK 293, HepG2, and MCF7 cells were then co-transduced with equal numbers of AAV2-Up and AAV2-Down viruses. In this experiment, HEK 293 is considered an easy-to-transfect cell line and HepG2 and MCF7 are considered hard-to-transfect cell lines. As a control, cells were transfected with a plasmid encoding Cas9 and a sgRNA targeting CCR5 by transfection. Seventy-two hours after either transfection or transduction, cells were harvested and analyzed using the Guide-it Mutation Detection Kit, a method that employs a mismatch specific endonuclease to identify insertions or deletions (indels). The resulting cleavage reaction was analyzed by agarose gel electrophoresis (Figure 5). The percentage of indels was quantified using densitometry (Cong et al. 2013). As anticipated, plasmid transfection of Cas9-sgRNA yielded significant indel formation only in HEK 293 cells. For the hard-to-transfect cells, HepG2 and MCF7 cells, there was almost no detectable indel formation (Figure 5). Conversely, AAV2-mediated delivery of Cas9-sgRNA complexes yielded significant indel formation in both HEK 293 and the hard-to-transfect cells (Figure 5). Interestingly, the fold difference in indel formation between transfection and transduction was far more pronounced in HepG2 and MCF7 cells than HEK 293 cells (5.6- and 6.3-fold versus 2.0-fold). These data underscore the efficacy of the AAVpro CRISPR/Cas9 Helper Free System (AAV2) at inducing site-specific genomic modifications in hard-to-transfect cells.
The AAVpro CRISPR/Cas9 Helper Free System (AAV2) is a complete system for the delivery of Cas9 and sgRNA to mammalian cells using AAV2. The kit contains all of the necessary reagents to prepare infection-ready, high-titer AAV particles to deliver Cas9 and a user-defined, gene-specific sgRNA. AAV-mediated delivery of Cas9 and sgRNA using this system, results in greater genome editing compared to plasmid transfection delivery, especially in hard-to-transfect cells.
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