CRISPR Activation (SAM-Mediated) Lentiviral Vector
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.
The Synergistic Activation Mediator (SAM) system is a powerful tool for transcriptional activation of genes within their endogenous genomic loci. This system is derived from CRISPR/Cas9 genome-editing systems, but rather than mediating genome editing, a modified type of gRNA directs the assembly of a multi-component transcriptional activation complex (SAM complex) at targeted sites. In general, assembly of the SAM complex is sufficient to induce very strong transcriptional activation of the target site.
The complete SAM system consists of three components, each provided in separate lentiviral vectors: The gRNA/MS2 expression vector, the MS2/P65/HSF1 helper vector, and the dCas9/VP64 helper vector.
The gRNA sequence selected by the user is cloned into the gRNA/MS2 expression vector. In this vector the gRNA is modified to include two 138-nt hairpin RNA aptamers which form binding sites for the bacteriophage MS2 coat proteins. These hairpin RNA aptamers linked to the gRNA facilitate the efficient recruitment of MS2-fusion proteins.
The MS2/P65/HSF1 helper vector drives expression of a three-domain fusion protein consisting of MS2, p65 (the trans-activation subunit of NF-kB), and HSF1 (the activation domain of human heat shock factor 1).
The dCas9/VP64 helper vector drives expression of a fusion protein consisting of a catalytically inactive variant of Cas9 and the synthetic VP64 transactivation domain.
When cells are co-transduced with these three vectors, the user-selected gRNA can potentially recruit both MS2/P65/HSF1 (via MS2-binding hairpin aptamers attached to the gRNA) and dCas9/VP64 (via CRISPR/Cas9 complex assembly) to gRNA target sites, thereby assembling powerful SAM complexes. These SAM complexes can achieve robust transcriptional activation of the target sites through synergistic interactions among the VP64, p65 and HSF1 activation domains.
This vector system is designed primarily for use in large-scale screens of genomic loci, using libraries of gRNA sequences to generate gRNA/MS2 expression vector libraries. However, this system can also be used to activate transcription of individual genes, or small sets of genes.
For further information about this vector system, please refer to the papers below.
Endogenous genomic context: The SAM system can activate transcription of target sites within their endogenous genomic loci. This is unlike transgenic or genome-editing methods which involve alterations to the genomic context of the gene of interest.
Orthogonal to physiological regulation: Targeted transcriptional activation of a gene using the SAM vector system does not require prior knowledge of how the gene of interest is naturally regulated. However, accurate DNA sequence information of the target site is necessary.
Strong activation: Transcriptional activation of genes using the SAM system can often achieve very high-level gene expression.
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.
Requires multiple vectors: This vector system requires co-expression of MS2/P65/HSF1 and dCas9/VP64 with gRNA/MS2, and separate vectors should be used for these components.
Specificity: The SAM based approach for targeted activation of genes is relatively new, and detailed information regarding the specificity of targeting using gRNA/MS2 RNAs is currently not available.
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 import 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: Allows in vitro transcription for RNA preparation. Scaffold gRNA sequence is included.
MS2 scaffold: This hairpin aptamer sequence binds robustly to fusion proteins containing the MS2 bacteriophage coat proteins.
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.
Design My Vector
Use this option to design your vector and
request cloning & downstream services.