In Vivo Testing
Lentivirus gRNA Expression Vector (Single gRNA)
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).
The lentivirus single gRNA expression vector system expresses a single guide RNA (gRNA) within transduced cells, which when coexpressed with the Cas9 nuclease, can lead to cleavage of the DNA sequence at a specific user-defined site in the genome. This vector is designed to be used together with another vector encoding the Cas9 nuclease.
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.
There are two commonly used variants of the Cas9 enzyme. The standard humanized Cas9 (hCas9) variant efficiently generates double-strand breaks (DSBs) at target sites. “Nickase” mutant form (Cas9_D10A) generate only single-stranded cuts in DNA, and if Cas9_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. This approach generally reduces off-target effects of CRISPR/Cas9 expression because targeting by both gRNAs is necessary for DSBs to be generated.
Cellular repair of DSBs by the nonhomologous end-joining pathway (NHEJ) usually results in small deletions, or more rarely insertions and base substitutions. When these mutations disrupt a protein-coding region (e.g. a deletion causing a frameshift), the result is a functional gene knockout. Alternatively, and less efficiently, DSBs can be repaired by homology-directed repair (HDR), using exogenous donor DNA template, which is co-introduced with the CRISPR/Cas9 vector. This can result in replacement of the target genomic DNA sequence with template sequence, generating small targeted base changes, such as point mutations. Nicked genomic DNA also frequently undergoes homology-directed repair (HDR), and if exogenous template DNA is introduced into the cell along with a targeted Cas9_D10A nickase, then small base changes can be generated.
Most DNA sequence can be effectively targeted using the CRISPR/Cas9 system. However, there is a strict requirement for an NGG (sometimes NAG) sequence, known as protospacer adjacent motif (PAM), which is located on the immediate 3’ end of the gRNA recognition sequence within the target DNA.
For further information on this vector system, please refer to the papers listed below.
|Science 339:819-23 (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. Biotech. 31:827–832 (2013)||Specificity of RNA-guided Cas9 nucleases|
|J Virol. 72:8463 (1998)||The 3rd generation lentivirus vectors|
|J Virol. 72:9873 (1998)||Self-inactivating lentivirus vectors|
|Science. 272:263 (1996)||Transduction of non-dividing cells by lentivirus vectors|
|Curr Gene Ther. 5:387 (2005)||Tropism of lentiviral vectors|
|J Virol. 77:4685 (2003)||Impact of cPPT on lentivirus vector transduction|
|Nat Protoc. 1:241 (2006)||Production and purification of lentiviral vectors|
Our Lentiviral CRISPR/Cas9 Expression Vectors are designed for quickly and efficiently expressing gRNAs for directing Cas9 nucleases to create small deletions at target sites in a cellular genome. To introduce mutations at a specific target site, a gRNA is chosen which matches the target DNA sequence. The nuclease must be expressed from another vector.
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, lentiviral transduction permanently inserts the user-designed DNA segments into host cells due to the integration of the viral vector into the host genome.
Simplicity: The simple homology relationship between the gRNA and the target makes the CRISPR/Cas9 system conceptually simple and easy to design.
High viral titer: Our lentiviral vector can be packaged into high titer virus. When lentivirus is obtained through our virus packaging service, the titer can reach >108 transducing unit per ml (TU/ml). At this titer, transduction efficiency for cultured mammalian cells can approach 100% when an adequate amount of viral 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).
Ability to concentrate viral particles: Our packaging system adds the VSV-G envelop protein to the viral surface. The structural robustness of this protein allows viral particles to be concentrated by high-speed centrifugation.
Relative uniformity of gene 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: While our vector is mostly used for in vitro transduction of cultured cells, it can also be used to transduce cells in live animals.
Effectiveness in vitro and in vivo: While our vector is mostly used for in vitro transduction of cultured cells, it can also be used to transduce cells 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 technical demanding and time consuming relative to conventional plasmid transfection.
Requires separate nuclease expression vector: Our lentiviral single gRNA vectors drive expression of a gRNA but not a Cas9 nuclease. A separate vector must be used along with this vector system in order to perform genome editing.
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. Off-target effects can be mitigated by using Cas9_D10A nickase in conjunction with two gRNAs (as described above), however this vector system includes only a single gRNA.
PAM requirement: CRISPR/Cas9 target sites must contain an NGG sequence, known as PAM, located on the immediate 3’ end of the gRNA recognition sequence.
RSV promoter: Rous sarcoma virus promoter. It drives transcription of viral RNA in packaging cells. This RNA is then packaged into live virus.
Δ5' LTR: 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: This drives high level expression of the gRNA.
gRNA: Allow in vitro transcription for RNA preparation. Scaffold gRNA sequence is included.
Terminator: Terminates transcription of the gRNA.
hPGK promoter: Human phosphoglycerate kinase 1 gene promoter. It drives the ubiquitous expression 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 transcriptional termination in the 3' LTR during viral RNA transcription, which leads to higher levels of functional viral RNA in packaging cells and hence greater viral titer. It also enhances transcriptional termination during the transcription of the user's gene of interest on the vector, leading to their higher expression levels.
ΔU3/3' LTR: 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 (due to the fact that 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.