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 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 AAV CRISPR vector helps to circumvent the mentioned challenges by expressing Cas9 and the desired gRNA sequence using a single AAV vector.
Our AAV CRISPR vector is designed to work with SaCas9 derived from Staphylococcus aureus, which is >1 kb shorter in comparison to the conventional SpCas9 derived from Streptococcus pyogenes. SaCas9 provides a distinct advantage over SpCas9 which has limited use in AAV-based applications due to its large size and the small cargo capacity of AAV vectors. SaCas9 functionally differs from SpCas9 in two major aspects – first, SaCas9 requires a different gRNA scaffold sequence from the one required by SpCas9. The gRNA compatible with SaCas9 is called as SagRNA. Secondly, the PAM sequence recognized by SaCas9 is NNGRR (NNGRRT preferred), whereas the PAM recognized by SpCas9 is NGG (preferred) and NAG (less preferred).
An AAV CRISPR 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 gRNA and Cas9 expression cassette placed in-between the two ITRs is introduced into target cells along with the rest of viral genome. A human U6 promoter drives the expression of the user-selected gRNA sequence, which directs Cas9 to the DNA target site of interest.
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 express genes on ssAAV vectors in host cells, 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.
AAV genomic DNA forms episomal concatemers in the host cell nucleus. In non-dividing cells, these concatemers can remain for the life of the host cells. In dividing cells, AAV DNA is lost through the dilution effect of cell division, because the episomal DNA does not replicate alongside host cell DNA. Random integration of AAV DNA into the host genome can occur but is extremely rare. This is desirable in many gene therapy settings where the potential oncogenic effect of vector integration can pose a significant concern.
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. The table below lists different AAV serotypes and their tissue tropism.
||Smooth muscle, CNS, brain, lung, retina, inner ear, pancreas, heart, liver
||Smooth muscle, CNS, brain, liver, pancreas, kidney, retina, inner ear, testes
||Smooth muscle, liver, lung
||CNS, retina, lung, kidney, heart
||Smooth muscle, CNS, brain, lung, retina, heart
||Smooth muscle, heart, lung, pancreas, adipose, liver
||Lung, liver, inner ear
||Smooth muscle, retina, CNS, brain, liver
||Smooth muscle, CNS, brain, retina, inner ear, liver, pancreas, heart, kidney, adipose
||Smooth muscle, lung, liver, heart, pancreas, CNS, retina, inner ear, testes, kidney, adipose
||Smooth muscle, lung, liver, heart, pancreas, CNS, retina, kidney
||Liver, heart, kidney, spleen
||Liver, brain, spleen, kidney
||Retina, inner ear
||Recommended AAV serotypes
||AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10
||AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV-PHP.eB
||AAV1, AAV2, AAV5, AAV7, AAV8, AAV-DJ/8
||AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV2.7m8
||AAV1, AAV2, AAV6.2, AAV8, AAV9, AAV2.7m8
||AAV1, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV9, AAVrh10
||AAV1, AAV2, AAV3, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ/8
||AAV1, AAV2, AAV6, AAV8, AAV9, AAVrh10
||AAV1,AAV4, AAV5, AAV6, AAV8, AAV9, AAVrh10, AAV-DJ
||AAV2, AAV4, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ/8
||AAV6, AAV8, AAV9
For further information about this vector system, please refer to the papers below.