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.