Adenovirus CRISPR Vector

CRISPR/Cas9 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 an all-in-one vector for expressing Cas9 and gRNA is that it provides the opportunity to deliver all the required components for CRISPR-mediated gene editing to the cell using a single vector which is technically straight forward. Using separate vectors for expressing Cas9 and gRNA requires co-transduction of the target cells with two separate vectors which can be technically challenging since not all cells will be transduced with both gRNA and Cas9 vectors simultaneously. An alternative approach for using separate vectors is to transduce cells or organisms stably expressing high-level of Cas9 with the desired gRNA sequences. However, this method can be considerably time-consuming and labor intensive. Our all-in-one adenovirus CRISPR vector helps to circumvent the mentioned challenges by expressing Cas9 and the desired gRNA sequence from a single adenoviral vector.

The adenovirus CRISPR vector is a highly efficient viral vehicle for adenovirus-mediated introduction of both Cas9 and the target site-specific gRNA sequence into a variety of mammalian cell types, where the vector remains as episomal DNA without integration into the host genome. It is the preferred gene delivery system in vivo, often used in gene therapy and vaccination. 

An adenovirus CRISPR vector is first constructed as a plasmid in E. coli. The gRNA and Cas9 expression cassette is cloned between the two inverted terminal repeats (ITRs) during vector construction. A human U6 promoter drives the expression of the user-selected gRNA sequence, which directs Cas9 to the DNA target site of interest. The vector is then transfected into packaging cells, where the region of the vector between the ITRs is packaged into live virus. When the virus is added to target cells, the DNA cargo is delivered into cells where it enters the nucleus and remains as episomal DNA without integration into the host genome. The gRNA and Cas9 expression cassette placed in-between the two ITRs during vector construction is introduced into target cells along with the rest of viral genome.

Our adenovirus CRISPR vector is available for expressing either single-gRNA or dual-gRNAs. While the single-gRNA vector is widely used for conventional CRISPR genome editing applications such as generating single gene knockouts, dual-gRNA vectors are necessary for applications requiring simultaneous targeting of a pair of genomic sites. Examples of such applications include: 1) paired Cas9 nickase experiments where the “nickase” mutant form (hCas9-D10A) of hCas9 is used in conjunction with two gRNAs targeting the two opposite strands of a single target site to generate single strand cuts one on each strand, thereby leading to a DSB with increased targeting specificity than a single gRNA; 2) generating deletion of a fragment between two DSBs targeted by a pair of gRNAs; and 3) targeting two different genes simultaneously. While the single gRNA vector consists of a single human U6 promoter driving the target site-specific gRNA sequence in between the two ITRs, the dual gRNA vector consists of two consecutive U6 promoters driving the expression of gRNA sequences specific to two genomic target sites of interest.

By design, 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).

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

Simplicity: The simple homology relationship between the gRNA and the target makes the CRISPR/Cas9 system conceptually simple and easy to design. Our adenovirus CRISPR vector system is designed for delivering both Cas9 as well as the target site-specific gRNA sequence to mammalian cells. This provides the opportunity to deliver all the required components for CRISPR-mediated gene editing to the target cells using a single adenoviral vector which is technically straight forward and less time-consuming than using two separate vectors for Cas9 and gRNA delivery.

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 infectious units per ml (IFU/ml).

Broad tropism: Cells from commonly used mammalian species such as human, mouse and rat can be transduced with our vector, 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.

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 adenoviral 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.

Lower specificity: Some off-target activity has been reported for the CRISPR/Cas9 system, and in general the TALEN system has lower off-target activity than CRISPR/Cas9. However, off-target effects can be significantly mitigated by using the mutant hCas9-D10A nickase in conjunction with two gRNAs to target the two opposite strands of a single target site to generate single strand cuts one on each strand, thereby leading to a DSB with increased targeting specificity than a single gRNA used in conjunction with the wild type hCas9 nuclease.

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.

Single-gRNA adenovirus CRISPR vector

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.

U6 Promoter: This drives high level expression of the downstream gRNA. This is the promoter of the human U6 snRNA gene, an RNA polymerase III promoter which efficiently expresses short RNAs.

gRNA: Guide RNA compatible with Cas9 derived from Streptococcus pyogenes.

Terminator: Terminates transcription of the gRNA.

CBh promoter: Chicken beta-actin promoter. Drives expression of the downstream Cas9 nuclease.

Cas9 protein: The open reading frame of the Cas9 nuclease is placed here. 

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.