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-transduction of the target cells with two separate vectors which can be technically challenging since not all cells will be transduced with both gRNA and Cas9 vectors simultaneously. An alternative approach for using separate vectors is to transduce 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 lentivirus CRISPR vector helps to circumvent the mentioned challenges by expressing Cas9 and the desired gRNA sequence from a single lentiviral vector.

Our lentivirus CRISPR vector is a highly efficient viral vehicle for permanently introducing both Cas9 and the target site- specific gRNA sequence into difficult-to-transfect mammalian cells. A lentivirus CRISPR vector is first constructed as a plasmid in E. coli. The gRNA and Cas9 expression cassette is cloned between the two long terminal repeats (LTRs) 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. The vector is then transfected into packaging cells along with several helper plasmids. Inside the packaging cells, vector DNA located between the LTRs is transcribed into RNA, and viral proteins expressed by the helper plasmids further package the RNA into virus. Live virus is then released into the supernatant, which can be used to infect target cells directly or after concentration. When the virus is added to target cells, the RNA cargo is shuttled into cells where it is reverse transcribed into DNA and permanently integrated into the host genome, leading to the co-expression of Cas9 and the user-selected gRNA sequence.

Our lentivirus 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 LTRs, the dual gRNA vector consists of two consecutive U6 promoters driving the expression of gRNA sequences specific to two genomic target sites of interest.

By design, our lentiviral vectors lack the genes required for viral packaging and transduction (these genes are instead carried by helper plasmids used during virus packaging). As a result, virus produced from lentiviral vectors has the important safety feature of being replication incompetent (meaning that they can transduce target cells but cannot replicate in them).

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

Our lentivirus CRISPR vector is derived from the third-generation lentiviral vector system. This system is optimized for high copy number replication in E. coli, high-titer packaging of live virus, efficient viral transduction of a wide range of cells, and efficient vector integration into the host genome. The lentivirus 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.

Figure 1. Gene editing with the all-in-one lentivirus-based CRISPR system. (A) Lentivirus vectors expressing Cas9:T2A:Puro and EGFP-targeting or scramble gRNA were packaged into the corresponding lentiviral particles and transduced into HEK293T cells stably expressing EGFP at MOI 10 or 50. Antibiotic selection with puromycin (Puro) was performed to isolate positively transduced cells. (B) EGFP in the transduced cells was observed under microscopy (100X). (C) The gRNA-targeted region was PCR amplified from the genomic DNA of non-transduced cells (NC), cells transduced with scramble gRNA, gRNA [EGFP-1], or gRNA [EGFP-2]. The gene editing was confirmed using the T7E1 assay. (D) The EGFP positive cells were quantified using flow cytometry. (E) The ratios of EGFP positive cells were decreased to 28-32% or 8-14% after editing using lentivirus at MOI 10 or 50, respectively. ***P<0.001, gRNA [EGFP-1] vs gRNA [scramble], ^###P<0.001, gRNA [EGFP-2] vs gRNA [scramble], ANOVA with Dunnett’s post hoc test.

Simplicity: The simple homology relationship between the gRNA and the target makes the CRISPR/Cas9 system conceptually simple and easy to design. Our lentivirus 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 lentiviral vector which is technically straight forward and less time-consuming than using two separate vectors for Cas9 and gRNA delivery.

High viral titer: Our vector can be packaged into high-titer virus (>10⁹ TU/ml when virus is obtained through our virus packaging service). At this viral titer, transduction efficiency for cultured mammalian cells can approach 100% when an adequate amount of viral supernatant is used.

Very broad tropism: Our packaging system adds the VSV-G envelop protein to the viral surface. This protein has broad tropism. As a result, cells from all commonly used mammalian species (and even some non-mammalian species) can be transduced. Furthermore, almost any mammalian cell type can be transduced (e.g. dividing cells and non-dividing cells, primary cells and established cell lines, stem cells and differentiated cells, adherent cells and non-adherent cells). Neurons, which are often impervious to conventional transfection, can be readily transduced by our lentiviral vector. Lentiviral vectors packaged with our system have broader tropism than adenoviral vectors (which have low transduction efficiency for some cell types) or MMLV retroviral vectors (which have difficulty transducing non-dividing cells).

Relative uniformity of vector delivery: Generally, viral transduction can deliver vectors into cells in a relatively uniform manner. In contrast, conventional transfection of plasmid vectors can be highly non-uniform, with some cells receiving a lot of copies while other cells receiving few copies or none.

Effectiveness in vitro and in vivo: Lentiviral vector systems can be used effectively in cultured cells and in live animals.

Safety: The safety of our vector is ensured by two features. One is the partition of genes required for viral packaging and transduction into several helper plasmids; the other is self-inactivation of the promoter activity in the 5' LTR upon vector integration. As a result, it is essentially impossible for replication competent virus to emerge during packaging and transduction. The health risk of working with our vector is therefore minimal.

Technical complexity: The use of lentiviral vectors requires the production of live virus in packaging cells followed by the measurement of viral titer. These procedures are technically demanding and time consuming relative to conventional plasmid transfection.

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 version 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 lentivirus CRISPR vector

CMV promoter: Human cytomegalovirus immediate early promoter. It drives transcription of viral RNA in packaging cells. This RNA is then packaged into live virus.

5' LTR-ΔU3: A deleted version of the HIV-1 5' long terminal repeat. In wildtype lentivirus, 5' LTR and 3' LTR are essentially identical in sequence. They reside on two ends of the viral genome and point in the same direction. Upon viral integration, the 3' LTR sequence is copied onto the 5' LTR. The LTRs carry both promoter and polyadenylation function, such that in wildtype virus, the 5' LTR acts as a promoter to drive the transcription of the viral genome, while the 3' LTR acts as a polyadenylation signal to terminate the upstream transcript. On our vector, Δ5' LTR is deleted for a region that is required for the LTR's promoter activity normally facilitated by the viral transcription factor Tat. This does not affect the production of viral RNA during packaging because the promoter function is supplemented by the CMV promoter engineered upstream of Δ5' LTR.

Ψ: HIV-1 packaging signal required for the packaging of viral RNA into virus.

RRE: HIV-1 Rev response element. It allows the nuclear export of viral RNA by the viral Rev protein during viral packaging.

cPPT: HIV-1 Central polypurine tract. It creates a "DNA flap" that increases nuclear importation of the viral genome during target cell infection. This improves vector integration into the host genome, resulting in higher transduction efficiency.

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.

EFS Promoter: Human eukaryotic translation elongation factor 1 α1 short form.  Drives expression of the downstream Cas9 nuclease.

Cas9 protein: The open reading frame of the Cas9 nuclease is placed here. 

WPRE: Woodchuck hepatitis virus posttranscriptional regulatory element. It enhances viral RNA stability in packaging cells, leading to higher titer of packaged virus.

3' LTR-ΔU3: A truncated version of the HIV-1 3' long terminal repeat that deletes the U3 region. This leads to the self-inactivation of the promoter activity of the 5' LTR upon viral vector integration into the host genome (since 3' LTR is copied onto 5' LTR during viral integration). The polyadenylation signal contained in ΔU3/3' LTR serves to terminates all upstream transcripts produced both during viral packaging and after viral integration into the host genome.

SV40 early pA: Simian virus 40 early polyadenylation signal. It further facilitates transcriptional termination after the 3' LTR during viral RNA transcription during packaging. This elevates the level of functional viral RNA in packaging cells, thus improving viral titer.

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.