Regular Plasmid gRNA Expression Vector

CRISPR/Cas9 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 both Cas9 as well as 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 combinatorial usage of different gRNA expression vectors in conjunction with a variety of Cas9 variants (wild type nuclease, nickase, nuclease-dead) depending upon the user’s experimental goal. Additionally, using a separate gRNA only vector allows cells or organisms stably expressing high levels of Cas9 to be transfected with different gRNA sequences targeting either the same gene or different genes. This provides the opportunity for comparing the efficiencies of different gRNA sequences in parallel at CRISPR-mediated gene targeting in cells or organisms with comparable and high levels of Cas9 expression.

The regular plasmid gRNA expression vector is a highly efficient tool for conventional transfection-based delivery of target site-specific gRNA sequences into mammalian cells. Delivering plasmid vectors into mammalian cells by conventional transfection is one of the most widely used procedures in biomedical research. While a number of more sophisticated gene delivery vector systems have been developed over the years such as lentiviral vectors, adenovirus vectors, AAV vectors and piggyBac, conventional plasmid transfection remains the workhorse of gene delivery in many labs. This is largely due to its technical simplicity as well as good efficiency 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%).

Our regular plasmid gRNA expression vector is available for expressing either single-gRNA or dual-gRNAs. While the single-gRNA vector is widely used for conventional CRISPR genome editing such as generating single gene knockout, dual-gRNA vectors are necessary for applications requiring simultaneous targeting of a pair of genomic sites. Examples of such applications include: 1) paired Cas9 nickase experiments where the “nickase” mutant form (hCas9-D10A) of hCas9 is used in conjunction with two gRNAs targeting the two opposite strands of a single target site to generate single strand cuts one on each strand, thereby leading to a DSB with increased targeting specificity than a single gRNA; 2) generating deletion of a fragment between two DSBs targeted by a pair of gRNAs; and 3) targeting two different genes simultaneously. While the single gRNA vector consists of a single human U6 promoter driving the target site-specific gRNA sequence, the dual gRNA vector consists of two consecutive U6 promoters driving the expression of gRNA sequences specific to two genomic target sites of interest.

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

Our regular plasmid gRNA 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. The regular plasmid gRNA expression vector is designed to drive high-level constitutive transcription of a user-selected gRNA sequence from a human U6 promoter to achieve highly efficient CRISPR targeting when used in conjunction with Cas9 nuclease. This vector is available for expressing either single-gRNA or dual-gRNAs enabling users to target either one or two genomic target sites of interest depending upon their experimental goal.

Flexibity: Our regular plasmid gRNA expression vector can be used in conjunction with a variety of Cas9 variants (nuclease, nickase, nuclease-dead) depending upon the user’s experimental goal. Additionally, this vector is available for expressing either single-gRNA or dual-gRNAs enabling users to target either one or two genomic target 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 genes carried on the vector.

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.

Single-gRNA regular plasmid expression vector

U6 Promoter: Drives expression of the downstream gRNA sequence. This is the promoter of the human U6 snRNA gene, an RNA polymerase III promoter which efficiently expresses short RNAs.

gRNA: Guide RNA compatible with the Cas9 variant being used.

Terminator: Terminates transcription of the gRNA.

hPGK promoter: Human phosphoglycerate kinase 1 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.

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

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.