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 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 combinatorial usage of 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.
Our regular plasmid Cas9 expression vector can be delivered to mammalian cells by conventional transfection which is of the most widely used procedures in biomedical research and remains the workhorse of gene delivery in many labs. This is largely due to the technical simplicity as well as good efficiency of transfection in a wide range of cell types. A key feature of transfection with regular plasmid vectors is that it is transient, with only a very low fraction of cells stably integrating the plasmid in the genome (typically less than 1%).
We offer multiple variants of the most widely used SpCas9 derived from Streptococcus pyogenes in our Cas9 nuclease collection to facilitate your vector design on our online portal. These variants include - hCas9, the 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.
Click to view the details of our Cas9 collections
For further information about this vector system, please refer to the papers below.