Lentivirus gRNA Expression Vector

Overview

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 transduced 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 lentivirus gRNA expression vector is a highly efficient viral vehicle for permanent delivery of target site-specific gRNA sequences into a variety of mammalian cells. A lentivirus gRNA expression vector is first constructed as a plasmid in E. coli. The gRNA expression cassette consisting of a human U6 promoter driving the target site-specific gRNA sequence is cloned between the two long terminal repeats (LTRs) during vector construction. It 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 expression of the user-selected gRNA sequence.

Our lentivirus 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 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).

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
Cell. 154: 1380 – 9 (2013) Use of Cas9 D10A double nicking for increased specificity
Nat. Biotechnol. 32: 267 (2014) CRISPR/Cas9 targeting using lentiviral gRNA expressing vectors
Plos One. 12: e0187236 (2017) CRISPR/Cas9 vectors for dual gRNA expression

Highlights

Our lentivirus gRNA expression 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 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.

Advantages

Flexibity: Our lentivirus 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.

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.

Disadvantages

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.

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

Single-gRNA expression lentiviral vector

RSV promoter: Rous sarcoma virus 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 RSV 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: 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.

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.

Dual-gRNA expression lentiviral vector

RSV promoter: Rous sarcoma virus 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 RSV 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: 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 #1: The first guide RNA compatible with the Cas9 variant being used.

gRNA #2: The second 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.

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.

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