Adeno-Associated Virus (AAV) Gene Targeting Donor Vector

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.

Cas9-mediated cleavage of the DNA target site ultimately results in a double-strand break (DSB) which can then be repaired by either of the two following repair pathways – the non-homologous end joining (NHEJ) pathway or the homology directed repair (HDR) pathway. Cellular repair of DSBs by NHEJ is more common and usually results in small deletions, or more rarely insertions and base substitutions. When these mutations disrupt a protein-coding region (e.g. a deletion causing a frameshift), the result is a functional gene knockout. Alternatively, and less efficiently, DSBs can be repaired by homology-directed repair (HDR), using exogenous donor DNA template, which is co-introduced into cells with the CRISPR/Cas9 components. This can result in replacement of the target genomic DNA sequence with sequence from the donor DNA, generating either small targeted base changes, such as point mutations or large sequence alterations such as fragment knockin.

Our gene targeting donor vectors are highly efficient vehicles for delivering exogenous donor templates to achieve targeted insertion of reporters, fluorescent tags or other desired sequences at genomic sites of interest. The donor vector is designed to contain the desired insertion sequence flanked by upstream and downstream homology arms (homologous sequences upstream and downstream of the target site of interest). Efficient HDR targeting also requires the DSB introduced by Cas9 to be located within the proximity of the target site of insertion, ideally within 10-15 bp of the homologous arms. Additionally, when designing donor vectors for HDR, it is critical to either exclude or inactivate any PAM sequences in the repair template when present, to prevent Cas9 from disrupting the donor template or the edited genomic locus after it has been edited by HDR.

An AAV gene targeting donor vector is first constructed as a plasmid in E. coli. It is then transfected into packaging cells along with helper plasmids, where the region of the vector between the two inverted terminal repeats (ITRs) is packaged into live virus. The donor sequence and homology arms placed in-between the two ITRs are introduced into target cells along with the rest of viral genome. Cas9 and gRNA from at least one other vector facilitate a targeted DSB, allowing the donor sequence to be inserted into the host genome.

The wild-type AAV genome is a linear single-stranded DNA (ssDNA) with two ITRs forming a hairpin structure on each end. It is therefore also known as ssAAV. In order to insert DNA into the host genome, the ssDNA genome needs to first be converted to double-stranded DNA (dsDNA) through two pathways: 1) synthesis of second-strand DNA by the DNA polymerase machinery of host cells using the existing ssDNA genome as the template and the 3' ITR as the priming site; 2) formation of intermolecular dsDNA between the plus- and minus-strand ssAAV genomes. The former pathway is the dominant one.

A major practical advantage of AAV is that in most cases AAV can be handled in biosafety level 1 (BSL1) facilities. This is due to AAV being inherently replication-deficient, producing little or no inflammation, and causing no known human disease. Due to their low immunogenicity in host organisms, our AAV CRISPR vectors are the perfect tools for in vivo CRISPR-based applications.

Many strains of AAV have been identified in nature. They are divided into different serotypes based on different antigenicity of the capsid protein on the viral surface. Different serotypes can render the virus with different tissue tropism (i.e. tissue specificity of infection). When our AAV vectors are packaged into virus, different serotypes can be conferred to the virus by using different capsid proteins for the packaging. The serotypes currently offered by us for our AAV vector systems include - serotypes 1, 2, 3, 4, 5, 6, 6.2, 7, 8, 9, rh10, DJ, DJ/8, PHP.eB, PHP.S, AAV2-retro and AAV2-QuadYF. During cloning, ITRs from AAV2 are used, as this is common practice in the field and does not impact specificity. Packaging helper plasmids include a Rep/Cap plasmid, containing the replication genes from AAV2 and the capsid proteins for a chosen serotype to determine tropism. The table below lists different AAV serotypes and their tissue tropism.

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

Our gene targeting donor vectors are designed to achieve highly efficient HDR-mediated insertion of reporters, fluorescent tags or other desired sequence at genomic target sites of interest. The donor vector is designed with the desired insertion sequence flanked by target site specific upstream and downstream homologous sequences to facilitate efficient recombination following CRISPR generated DSBs at the genomic target sites. The donor sequence and homology arms are designed in an AAV transfer vector optimized for high-titer packaging into live virus and efficient transduction of host cells using all known capsid serotypes.

Precise changes:  Delivering exogenous repair templates in the form of gene targeting donor vectors enables HDR-mediated introduction of precise sequence changes at the genomic target sites of interest.

High viral titer:  Our AAV vector can be packaged into high titer virus. When AAV virus is obtained through our virus packaging service, titer can reach >10¹³ genome copy per ml (GC/ml).

Broad tropism: A wide range of cell and tissue types from commonly used mammalian species such as human, mouse and rat can be readily transduced with our AAV vector when it is packaged into the appropriate serotype. But some cell types may be difficult to transduce, depending on the serotype used (see disadvantages below).

Small cargo space:  AAV has the smallest cargo capacity of any of our viral vector systems. AAV can accommodate a maximum of 4.7 kb of sequence between the ITRs, which leaves ~4.2 kb cargo space for user's DNA of interest.

Difficulty transducing certain cell types:  Our AAV vector system can transduce many different cell types including non-dividing cells when packaged into the appropriate serotype. However, different AAV serotypes have tropism for different cell types, and certain cell types may be hard to transduce by any serotype.

Low efficiency:   Upon Cas9-induced cleavage of DNA target sites, HDR-mediated repair of the cleaved sites occurs at a much lower frequency than compared to NHEJ-mediated repair. As a result, CRISPR/Cas9 targeting in the presence of an exogenous donor template will give rise to a mixed population of cells, some repaired by the NHEJ pathway while others repaired by the HDR pathway. Therefore, careful screening of the resultant cell population is essential to isolate clones containing the desired HDR-edited sequence. For obtaining cells with homologous alleles altered in the same way, additional round(s) of knockin screen are often needed.

PAM requirement:   CRISPR/Cas9 target sites must contain an NGG sequence, known as PAM, located on the immediate 3’ end of the gRNA recognition sequence. It is critical to either exclude or inactivate any PAM sequences in the donor vector when present, to prevent Cas9 from disrupting the donor template or the edited genomic locus after it has been edited by HDR.

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.

5’-Homology Arm:  Homologous sequence immediately upstream of the target site of insertion. The length of the homology arms depends on the length of the sequence to be inserted, with longer insertions requiring longer arms.

Donor Sequence/Cassette: User-selected insertion sequence to be knocked in at the genomic target site of interest.

3’-Homology Arm: User-selected insertion sequence to be knocked in at the genomic target site of interest.

3' ITR: 3' inverted terminal repeat. See description for 5’ ITR.

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

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.