Regular Plasmid Cas9 Expression Vector

Overview

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.

References Topic
Science. 339:819 (2013) Description of genome editing using the CRISPR/Cas9 system
Nat. Biotech. 31:827 (2013) Specificity of RNA-guided Cas9 nucleases
Nat. Commun. 9:1911 (2018) Review on various Cas9 variants

Highlights

Our Cas9 only vectors are designed to drive high-level Cas9 expression under a user-selected promoter to achieve highly efficient CRISPR targeting when used in conjunction with target site-specific gRNA sequences. We offer a variety of Cas9 variants to help you select the right one suitable for your experimental design. Our regular plasmid Cas9 expression vector is optimized for high copy number replication in E. coli and high-efficiency transfection. Cells transfected with the vector can be selected and/or visualized based on marker gene expression as chosen by the user.

Advantages

Transient expression: Transfection of the Cas9 only vector results in strong transient expression of the Cas9 protein within the target cells. Without drug selection, the plasmid will be lost over time eliminating the Cas9 from the target cells after genome editing has taken place.

Flexibility: The Cas9 only vector can be co-transfected with multiple different gRNA sequences for targeting different genomic sites of interest.

Technical simplicity: Delivering plasmid vectors into cells by conventional transfection is technically straightforward, and far easier than virus-based vectors which require the packaging of live virus.

High-level expression: Conventional transfection of plasmids can often result in very high copy numbers in cells (up to several thousand copies per cell). This can lead to very high expression levels of the Cas9 variant carried by the vector.

Disadvantages

Non-integration of vector DNA: Conventional transfection of plasmid vectors is also referred to as transient transfection because the vector stays mostly as episomal DNA in cells without integration. However, plasmid DNA can integrate permanently into the host genome at a very low frequency (one per 102 to 106 cells depending on cell type). If a drug resistance or fluorescence marker is incorporated into the plasmid, cells stably integrating the plasmid can be derived by drug selection or cell sorting after extended culture.

Limited cell type range: The efficiency of plasmid transfection can vary greatly from cell type to cell type. Non-dividing cells are often more difficult to transfect than dividing cells, and primary cells are often harder to transfect than immortalized cell lines. Some important cell types, such as neurons and pancreatic β cells, are notoriously difficult to transfect. Additionally, plasmid transfection is largely limited to in vitro applications and rarely used in vivo.

Non-uniformity of gene delivery: Although a successful transfection can result in very high average copy number of the transfected plasmid vector per cell, this can be highly non-uniform. Some cells can carry many copies while others carry very few or none. This is unlike transduction by virus-based vectors which tends to result in relatively uniform gene delivery into cells.

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. The required PAM sequence varies depending on the Cas9 variant being used.

Key components

Promoter: The promoter that drives the expression of the downstream Cas9 gene is placed here.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

ORF: The open reading frame of the Cas9 nuclease variant chosen by the user.

SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

CMV promoter: Human cytomegalovirus immediate early promoter. It drives the ubiquitous expression of the downstream marker gene.

Marker: A drug selection gene (such as neomycin resistance), a visually detectable gene (such as EGFP), or a dual-reporter gene (such as EGFP/Neo). This allows cells transduced with the vector to be selected and/or visualized.

BGH pA: Bovine growth hormone polyadenylation. It facilitates transcriptional termination of the upstream ORF.

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

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

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