Adenovirus Cas9 Expression Vector

Overview

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) nuclease expression vectors are among several types of emerging genome editing tools that can quickly and efficiently create mutations at target sites of a genome (the other two popular ones being ZFN and TALEN).

Cas9 is a member of a class of RNA-guided DNA nucleases which are part of a natural prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and bacteriophage. Within the cell, the Cas9 enzyme forms a complex with a guide RNA (gRNA), which provides targeting specificity through direct interaction with homologous 18-22nt target sequences in the genome. Hybridization of the gRNA to the target site localizes Cas9, which then cuts the target site in the genome.

To achieve CRISPR-mediated gene targeting it is essential for the target cells to co-express Cas9 and the target site-specific gRNA at the same time. This can be accomplished by either expressing both Cas9 and the gRNA sequence from the same vector (a.k.a. all-in-one vector) or by using separate vectors for driving Cas9 and gRNA expression (Cas9 only and gRNA only vectors, respectively). The advantage of using separate vectors over an all-in-one vector for expressing Cas9 and gRNA is that it offers the flexibility of using different gRNA expression vectors in conjunction with a variety of Cas9 variants (wild type nuclease, Cas9 nickase, dCas9, etc.) depending upon the user’s experimental goal.

The adenovirus Cas9 expression vector system is an efficient method for achieving Cas9 expression in many (but not all) mammalian cell types where the vector remains as episomal DNA in cells, without integrating into the host genome. It is the preferred gene delivery system in vivo, often used in gene therapy and vaccination.

An adenovirus Cas9 expression vector is first constructed as a plasmid in E. coli, and is then transfected into packaging cells, where the region of the vector between the two inverted terminal repeats (ITRs) is packaged into live virus. After the viral genome is delivered into target cells, it enters the nucleus and remains as episomal DNA. The Cas9 gene placed in-between the two ITRs during vector construction is introduced into target cells along with the rest of viral genome.

By design, our adenoviral vectors lack the E1A, E1B and E3 genes (delta E1 + delta E3). The first two are required for the production of live virus (these two genes are engineered into the genome of packaging cells). As a result, virus produced from the vectors have the important safety feature of being replication incompetent (meaning that they can transduce target cells but cannot replicate in them).

We offer multiple variants of the most widely used SpCas9 derived from Streptococcus pyogenes, to help you find the right Cas9 suitable for your experimental design. These include - hCas9, the standard humanized version of wild type SpCas9 which efficiently generates double-strand breaks (DSBs) at target sites; hCas9-D10A, the “nickase” mutant form of hCas9 which generates only single-stranded cuts in DNA; dCas9, a catalytically inactive variant of SpCas9, bearing both D10A and H840A mutations; SpCas9-HF1, a high-fidelity variant of SpCas9; and eSpCas9, an enhanced specificity variant of SpCas9. Fusions of dCas9 with activation domains such as dCas9/VP64 and dCas9/VPR or with repression domains such as dCas9/KRAB are also available for CRISPRa and CRISPRi applications respectively. Additionally, we offer SaCas9 derived from Staphylococcus aureus for applications requiring a shorter Cas9 variant compared to Spcas9 and AsCpf1 derived from Acidaminococcus for achieving DNA cleavage via staggered DNA double stand breaks.

For further information about this vector system, please refer to the papers below.

References Topic
Science. 339:819 (2013) Description of genome editing using the CRISPR/Cas9 system
Nat. Biotech. 31:827 (2013) Specificity of RNA-guided Cas9 nucleases
Nat. Commun. 9:1911 (2018) Review on various Cas9 variants
Sci Rep. 4:5105 (2014) CRISPR/Cas9 targeting by adenoviral vectors

Highlights

Our adenovirus Cas9 expression vector is derived from the adenovirus serotype 5 (Ad5). It is optimized for high copy number replication in E. coli, high-titer packaging of live virus and efficient transduction of host cells. The adenovirus Cas9 expression vectors are designed to drive high-level Cas9 expression under a user-selected promoter to achieve highly efficient CRISPR targeting when used in conjunction with DNA target site-specific gRNA sequences. We offer a variety of Cas9 variants to help you select the right one suitable for your experimental design.

Advantages

Flexibility: The Cas9 only vector can be co-transduced with multiple different gRNA sequences for targeting different genomic sites of interest.

Low risk of host genome disruption: Upon transduction into host cells, adenoviral vectors remain as episomal DNA in the nucleus. The lack of integration into the host genome can be a desirable feature for in vivo human applications, as it reduces the risk of host genome disruption that might lead to cancer.

Very high viral titer: After our adenoviral vector is transfected into packaging cells to produce live virus, the virus can be further amplified to very high titer by re-infecting packaging cells. This is unlike lentivirus, MMLV retrovirus, or AAV, which cannot be amplified by re-infection. When adenovirus is obtained through our virus packaging service, titer can reach >1011 plaque-forming unit per ml (PFU/ml).

Broad tropism: Cells from commonly used mammalian species such as human, mouse and rat can be transduced with our adenoviral vectors. But some cell types have proven difficult to transduce (see disadvantages below).

Effectiveness in vitro and in vivo: Our vector is often used to transduce cells in live animals, but it can also be used effectively in vitro.

Safety: The safety of our vector is ensured by the fact that it lacks genes essential for virus production (these genes are engineered into the genome of packaging cells). Virus made from our vector is therefore replication incompetent except when it is used to transduce packaging cells.

Disadvantages

Non-integration of vector DNA: The adenoviral genome does not integrate into the genome of transduced cells. Rather, it exists as episomal DNA, which can be lost over time, especially in dividing cells.

Difficulty transducing certain cell types: While our adenoviral vectors can transduce many different cell types including non-dividing cells, it is inefficient against certain cell types such as endothelia, smooth muscle, differentiated airway epithelia, peripheral blood cells, neurons, and hematopoietic cells.

Strong immunogenicity: Live virus from adenoviral vectors can elicit strong immune response in animals, thus limiting certain in vivo applications.

Technical complexity: The use of viral vectors requires the production of live virus in packaging cells followed by the measurement of viral titer. These procedures are technical demanding and time consuming relative to conventional plasmid transfection. These demands can be alleviated by choosing our virus packaging services when ordering your vector.

PAM requirement: CRISPR/Cas9 based targeting is dependent on a strict requirement for a protospacer adjacent motif (PAM), located on the immediate 3’ end of the gRNA recognition sequence. The required PAM sequence varies depending on the Cas9 variant being used.

Key components

5' ITR: 5' inverted terminal repeat. In wild type virus, 5' ITR and 3' ITR are essentially identical in sequence. They reside on two ends of the viral genome pointing in opposite directions, where they serve as the origin of viral genome replication.

Ψ: Adenovirus packaging signal required for the packaging of viral DNA into virus. 

Promoter: The promoter that drives the expression of the downstream Cas9 gene is placed here.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

ORF: The open reading frame of the Cas9 nuclease variant chosen by the user.

TK pA: Herpes simplex virus thymidine kinase polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

ΔAd5: Portion of Ad5 genome between the two ITRs minus the E1A, E1B and E3 regions.

3' ITR: 3' inverted terminal repeat.

pBR322 ori: pBR322 origin of replication. Plasmids carrying this origin exist in medium copy numbers in E. coli.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

PacI: PacI restriction site (PacI is a rare cutter that cuts at TTAATTAA). The two PacI restriction sites on the vector can be used to linearize the vector and remove the vector backbone from the viral sequence, which is necessary for efficient packaging.

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