PiggyBac 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 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. Additionally, using a separate Cas9 vector allows generation of stable cell lines with high levels of Cas9 expression which can then be transfected with the desired gRNA sequences. This approach can help to achieve better targeting performance than compared to an all-in-one CRISPR vector due to uniform and high-level of Cas9 expression in all cells.

Our piggyBac Cas9 expression vector system is a simple and efficient method for permanently introducing Cas9 into a wide variety of mammalian cell types. It is a transposon-based system that utilizes plasmid transfection (rather than viral transduction) to permanently integrate a Cas9 expression cassette into the host cell genome.  The piggyBac Cas9 expression vector system contains two vectors, both engineered as E. coli plasmids. One vector, referred to as the helper plasmid, encodes the transposase. The other vector, referred to as the transposon plasmid, contains two inverted terminal repeats (ITRs) bracketing the region to be transposed, which includes the Cas9-expression cassette.

When the helper and transposon plasmids are co-transfected into target cells, the transposase produced from the helper plasmid recognizes the two ITRs on the transposon, and inserts the flanked region including the two ITRs into the host genome. PiggyBac is a class II transposon, meaning that it moves in a cut-and-paste manner, hopping from place to place without leaving copies behind. (In contrast, class I transposons move in a copy-and-paste manner.) Because the helper plasmid is only transiently transfected into host cells, it will get lost over time. With the loss of the helper plasmid, the integration of the Cas9-expressing transposon in the genome of host cells becomes permanent. If these cells are transfected with the helper plasmid again, the transposon could get excised from the genome of some cells, footprint free.

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.

References Topic
Science. 339:819 (2013) Description of genome editing using the CRISPR/Cas9 system
Nat. Biotech. 31:827 Specificity of RNA-guided Cas9 nucleases
Nat. Commun. 9:1911 (2018) Review on various Cas9 variants
Genome Res. 24:1526 (2014) PiggBac vector based CRISPR/Cas9 editing

Highlights

Our piggyBac transposon vector along with the helper plasmid are optimized for high copy number replication in E. coli, and efficient transfection into a wide range of target cells. Our piggyBac Cas9 expression vectors are designed to drive high-level permanent Cas9 expression under a user-selected promoter to achieve highly efficient CRISPR targeting when used in conjunction with DNA target site-specific gRNA sequences. We offer a variety of Cas9 variants to help you select the right one suitable for your experimental design.

Advantages

Permanent integration of vector DNA: Conventional transfection results in almost entirely transient delivery of DNA into host cells due to the loss of DNA over time. This problem is especially prominent in rapidly dividing cells. In contrast, transfection of mammalian cells with the piggyBac transposon plasmid along with the helper plasmid can deliver genes carried on the transposon permanently into host cells due to the integration of the transposon into the host genome.

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.

Very large cargo space: Our transposon vector can accommodate ~30 kb of total DNA. The plasmid backbone and transposon-related sequences only occupy about 3 kb, leaving plenty of room to accommodate any of the user-selected Cas9 variants along with appropriate selection markers.

Disadvantages

Limited cell type range: The delivery of the piggyBac Cas9 expression vector into cells relies on transfection. The efficiency of 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. These issues limit the use of the piggyBac system.

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

5' ITR: 5' inverted terminal repeat. When a DNA sequence is flanked by two ITRs, the piggyBac transposase can recognize them, and insert the flanked region including the two ITRs into the host genome. 

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.

rBG pA: Rabbit β-globin 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 signal. It facilitates transcriptional termination of the upstream ORF.

3' ITR: 3' inverted terminal repeat.

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.