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 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 an all-in-one vector for expressing Cas9 and gRNA is that it provides the opportunity to deliver all the required components for CRISPR-mediated gene editing to the cell using a single vector which is technically straight forward. Using separate vectors for expressing Cas9 and gRNA requires co-transfection of the target cells with two separate vectors which can be technically challenging since not all cells will be transfected with both gRNA and Cas9 vectors simultaneously. An alternative approach for using separate vectors is to transfect cells or organisms stably expressing high-level of Cas9 with the desired gRNA sequences. However, this method can be considerably time-consuming and labor intensive. Our all-in-one piggyBac CRISPR vector helps to circumvent the mentioned challenges by expressing Cas9 and the desired gRNA sequence from a single piggyBac vector.

Our piggyBac CRISPR vector is highly effective for achieving transfection-mediated permanent introduction of both Cas9 and the target site-specific gRNA sequence into the host genome of mammalian cells. The piggyBac 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 terminal repeats (TRs) bracketing the region to be transposed. The gRNA and Cas9 expression cassette is cloned into this region during vector construction. A human U6 promoter drives the expression of the user-selected gRNA sequence, which directs Cas9 to the DNA target site of interest. When the helper and transposon plasmids are co-transfected into target cells, the transposase produced from the helper would recognize the two TRs on the transposon, and insert the flanked region including the two TRs into the host genome. Insertion typically occurs at host chromosomal sites that contain the TTAA sequence, which is duplicated on the two flanks of the integrated fragment.

Our piggyBac CRISPR vector is available for expressing either single-gRNA or dual-gRNAs. While the single-gRNA vector is widely used for conventional CRISPR genome editing applications such as generating single gene knockouts, 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 in between the two TRs, the dual gRNA vector consists of two consecutive U6 promoters driving the expression of gRNA sequences specific to two genomic target sites of interest.

Two variants of Cas9 enzyme are available in our all-in-one piggyBac CRISPR vectors. The standard humanized Cas9 (hCas9) variant efficiently generates double-strand breaks (DSBs) at target sites, while the “nickase” mutant form (hCas9-D10A) generates only single-stranded cuts in DNA. If hCas9-D10A nickase is used in conjunction with two gRNAs targeting the two opposite strands of a single target site, then the nickase enzyme will generate single strand cuts on both strands, resulting in DSBs at the target site (as described above). This approach generally reduces off-target effects of CRISPR/Cas9 expression because targeting by both gRNAs is necessary for DSBs to be generated.

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 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.

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

Our piggyBac CRISPR vector along with the helper plasmid are optimized for high copy number replication in E. coli, efficient transfection into a wide range of target cells, and high-level expression of the transgene carried on the vector. The piggyBac CRISPR vector system is designed to deliver Cas9 and a target site-specific gRNA sequence using a single vector. 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.

Simplicity: The simple homology relationship between the gRNA and the target makes the CRISPR/Cas9 system conceptually simple and easy to design. Our piggyBac CRISPR vector system is designed for delivering both Cas9 as well as the target site-specific gRNA sequence to mammalian cells. This provides the opportunity to deliver all the required components for CRISPR-mediated gene editing to the target cells using a single piggyBac vector which is technically straight forward and less time-consuming than using two separate vectors for Cas9 and gRNA delivery.

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.

Limited cell type range: The delivery of piggyBac vectors 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.

Lower specificity: Some off-target activity has been reported for the CRISPR/Cas9 system, and in general the TALEN system has lower off-target activity than CRISPR/Cas9. However, off-target effects can be significantly mitigated by using the mutant hCas9-D10A nickase in conjunction with two gRNAs to target 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 used in conjunction with the wild type hCas9 nuclease.

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. 

Single-gRNA piggyBac CRISPR vector

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

U6 Promoter: This drives high level expression of the downstream gRNA. 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 Cas9 derived from Streptococcus pyogenes.

Terminator: Terminates transcription of the gRNA.

CBh promoter: Chicken beta-actin promoter. Drives expression of the downstream Cas9 nuclease.

Cas9 protein: Cas9 variant chosen by 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 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.