AAV Plasmid Design and Construction

PackGene’s proprietary AAV Cas9 expression vectors have been carefully designed to co-express SpCas9 or SaCas9, sgRNA, or both Cas9 and sgRNA from a single vector.

Available in piVector Designer

• SpCas9: dual vector or all-in-one vector
  PackGene’s proprietary SpCas9 expression vectors are engineered to co-express SpCas9 and sgRNA within a single AAV vector. To ensure that the SpCas9 expression sequence stays within the AAV vector insert length limit of ~5Kb, we use the compact and efficient promoter sequences EF1s or miniCMV. For sgRNA expression, we use the efficient and relatively short H1 promoter to reduce the total bp length of the sgRNA expression cassette. With this strategy, we have generated a single AAV CRISPR vector capable of reliable and efficient SpCas9 and sgRNA co-expression, minimizing the risk of mixed cell expression phenotypes and the potential for inconsistent or spurious data.
• SaCas9: dual vector or all-in-one vector
  SaCas9 is a shorter version of Cas9 that maintains similar cleavage activity to SpCas9 but is encoded by a shorter gene of approximately 3.2 kb compared to SpCas9’s ~4.2 kb. In addition, the SaCas9 PAM sequence (NNGRRT) occurs less frequently in host genomes, approximately once every 32 bp, and the 21-23 nt target sequence is longer than that of traditional SpCas9. This reduced PAM frequency and extended target sequence length contribute to SaCas9’s higher binding specificity, theoretically resulting in a lower off-target cleavage rate compared to traditional SpCas9.

Available upon request, please contact our technical support team

• SpCas9HF
  Hi-fidelity version of SpCas9
• SaCas9HF
  Hi-fidelity version of SaCas9
• AAV-CRISPR gene activation MS2-P65-HSF1
  While Cas9 is typically paired with traditional sgRNA to produce targeted gene knockouts, it can also be paired with a sgRNA containing a modified MS2 RNA linker scaffold for an alternative function. In the modified MS2 configuration, SpCas9 loses its DNA cleavage capability and instead recruits the MS2-P65-HSF1 transcriptional activation complex, which drives downstream gene transcription. This approach enables transcriptional activation, potentially increasing endogenous gene expression by more than 1000-fold.
• NmCas9
  The 3.3-kb-long NmCas9 is an alternative version of Cas9 with similar activity to SpCas9 but offers an advantage in applications where exceptionally low off-target cleavage rates are required. NmCas9 recognizes a longer PAM sequence (NNNNGATT), which occurs less frequently than the SpCas9 PAM at approximately once every 128 bp. Combined with its extended target sequence length of 21-23 nt, this results in higher target specificity. These characteristics contribute to NmCas9’s theoretically lower off-target cleavage rate compared to other Cas9 variants, making it ideal for highly precise genome editing applications.
• AsCpf1 and LbCpf1
  While Cas9 and its variants are the most commonly used CRISPR effector endonucleases, the Cpf1 endonuclease has gained increasing popularity as an alternative for CRISPR gene editing. Similar to Cas9, Cpf1 requires a gRNA for activation, binds genomic DNA adjacent to a PAM sequence, and cleaves DNA following sgRNA target sequence annealing. However, Cpf1 is shorter in size than Cas9, and requires a shorter sgRNA for full functionality. Another key distinction is that while Cas9 generates blunt-ended DNA cuts, Cpf1 produces 4-5 base pair protruding sticky ends, facilitating low-error and controllable gene insertions.

What is CRISPR?
CRISPR is a state-of-the-art genetic modification tool that can be used to knockout, mutate, or overexpress a gene of interest. CRISPR-based research strategies have been widely employed across biological fields of study due to their powerful performance and relatively straightforward mechanism of action.

CRISPR gene editing requires three components: (1) a guide RNA (gRNA), (2) a Cas9 endonuclease, and (3) a protospacer adjacent motif (PAM) within the target genome. The gRNA is a single stranded RNA molecule consisting of two regions: (1) a gene-targeting sequence that complements a 15-24 base pair segment of the gene of interest, and (2) a scaffold region that binds to Cas9. The gRNA scaffold region binds to a Cas9 endonuclease while leaving the host DNA compliment region of the gRNA unbound. The Cas9-gRNA complex may then scan the host cell’s genomic DNA for a specific 2-6 base pair sequence referred to as the PAM. Once the Cas9-gRNA complex recognizes the PAM sequence the gene targeting region of the gRNA may then anneal to the host DNA adjacent to the PAM sequence. After successful binding of the gRNA gene targeting region Cas9 will cleave the host DNA adjacent to the PAM sequence.

Cas9 mediated DNA cleavage generally results in gross disruptions in gene transcription and functional gene knockout following DNA repair. Alternatively, cleavage can be used to introduce precise DNA mutations or to activate gene expression using a modified gRNA scaffold region that inhibits Cas9 cleavage capabilities while simultaneously recruiting transcriptional proteins to a gene’s promoter region. Importantly, the specificity of target DNA recognition makes it feasible to design CRISPR based gene editing strategies targeted at nearly any gene.

The design and production of AAV vectors for CRISPR based gene editing can be both challenging and time-consuming. However, our professional team has experience and knowledge necessary to deliver high quality CRISPR-AAVs that are ready to use for your experimental needs immediately on delivery.

PackGene provides AAV vectors for reducing protein translation via short-hairpin RNA (shRNA), a widely used RNA interference (RNAi) technique. We offer options to express shRNA under the H1 or U6 promoter, and all of our AAV-shRNA vectors are designed to co-express a reporter protein of your choosing. Additionally, PackGene provides expert assistance with AAV shRNA cloning and vector construction. Please inquire for more details.
Available in piVector Designer

Pol III Promoter driven shRNA | miR30-Based shRNA

RNA interference (RNAi) is a highly specific tool for protein knockdown, where small non-coding RNAs bind to a target proteins mRNA, promoting its degradation and consiquently reducing protein translation. RNAi mechanisms are endogenously present in most eukaryotes, and have been widely adopted as a molecular tool for gene function studies, drug discovery, and gene silencing therapy.

Recent clinical trials have demonstrated that RNAi hold significant potential in targeting disease-causing genes for the treatment of human diseases. In comparison to other delivery methods, AAV vector-based expression of RNAi has been shown to drive prolonged reduction of mRNA targets in live animals.

PackGene offers custom inhibitory miRNA AAV vectors designed to inhibit endogenous miRNA expression and function in live animals. AAV-miRNA cloning ensures greater expression durability and longer effect duration compared to traditional synthetic miRNA inhibitors while also improving safety and minimizing immunogenicity.

miRNA are small non-coding RNAs that are endogenously expressed in eukaryotes and play important role in regulating gene expression. miRNA inhibitors are RNA molecules that are designed as reverse compliment sequence to endogenous miRNA and functionally block their gene regulation capabilities.

Our service offers direct, convenient, and efficient expression of miRNA inhibitors for in vivo animal experiments, allowing for targeted delivery to specific organs and tissues via custom AAV serotypes. This approach ensures greater expression durability and longer effect duration compared to traditional synthetic miRNA inhibitors, while also increasing safety and reducing immunogenicity.