gRNA and Cas9 Coexpression
CRISPR-Based Gene Activation
Regular Plasmid gRNA Sensor Vector (for Testing gRNA Specificity)
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). These plasmid vectors encode a sequence-specific RNA-guided DNA nuclease (or nickase) enzyme, which can be used to edit the DNA sequence of specific user-defined sites in the genome.
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 gRNA sensor vector system is designed for testing the cleavage efficiency of CRISPR/Cas9 at one or multiple gRNA target sequences. This system allows users to identify site(s) that are the most effective CRISPR/Cas9 targets by comparing multiple gRNA target sites.
The basis for the gRNA sensor vector system is an inactive, split EGFP ORF, placed downstream of the strong ubiquitous promoter, EF1A. The gRNA target site to be tested is cloned between the two incomplete, nonfunctional sections of the EGFP ORF (termed EGFP-L and EGFP-R). While EGFP-L consists of the first 468 bp of the EGFP ORF, EGFP-R corresponds to base pairs 268-720 of the EGFP ORF. The 200 bp from the 3’end of EGFP-L share sequence homology with the 200 bp from the 5’end of EGFP-R.
When the gRNA sensor vector is transfected into cells alone, no green fluorescence is observed since neither EGFP-L nor EGFP-R encodes functional fluorescent protein. However, if the gRNA sensor vector is co-transfected into cells along with Cas9 and the corresponding gRNA vectors, the gRNA-Cas9 complex would be recruited to the gRNA target sequence between EGFP-L and EGFP-R, thereby cutting it and generating a double-stranded break (DSB). This DSB would allow homologous recombination to occur between the homologous regions of EGFP-L and EGFP-R. Very often, the homologous recombination will result in an intact functional EGFP ORF, producing green fluorescent protein. In this system, multiple gRNA target sites (each with PAM sequence) can be cloned in tandem between EGFP-L and EGFP-R, and can be tested individually by co-transfecting with Cas9 and the corresponding gRNA. Analysis of the efficiency at which EGFP fluorescent cells are generated allows quantitative comparison of CRISPR/Cas9 cleavage efficiency at different gRNA target sites.
For further information about this vector system, please refer to the paper below.
|Appl Biochem Biotechnol. 180:655 (2016)||Description of gRNA sensor system|
This vector system is designed for the quantitative comparison of cleavage efficiency of CRISPR/Cas9 at gRNA target sites in mammalian cells by observation of EGFP fluorescence. The regular plasmid system is technically simple and straightforward to use.
Simplified testing system: Analysis of gRNA target site cleavage efficiency using this system is straightforward, simple and fast.
Sensitive and robust readout: The strength of the EF1A promoter and the robustness of EGFP fluorescence allows detection of fluorescence even in cells with very low copy numbers.
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.
Simplified system: In this sensor vector system, the gRNA target sequences are not in the same genetic context as within the genome of interest, and the sensor vector may be present in cells at high copy number when testing is performed. CRISPR/Cas9 cleavage of gRNA target sites may have somewhat altered efficiency in this assay system.
No off-target information: This system only tells you the efficiency of CRISPR/Cas9 cleavage on gRNA target sites, without providing any information on off-target effects.
EF1A promoter: Human eukaryotic translation elongation factor 1 α1 promoter. A strong, constitutive promoter driving the expression of EGFP-L:gRNA Target:EGFP-R cassette.
Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest to facilitate translation initiation in eukaryotes.
EGFP-L: Corresponds to positions 1-468 bp of the EGFP ORF. This does not encode a functional fluorescent protein.
gRNA Target Sequence: The gRNA target sequence to be tested is cloned here. Multiple gRNA target sequences can be cloned here in tandem. PAM sequence must be included.
EGFP-R: Corresponds to positions 268-720 bp of the EGFP ORF. This does not encode a functional fluorescent protein.
SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.
Marker: A drug selection gene (such as neomycin resistance), a visually detectable gene (such as mCherry, should avoid green fluorescent protein), or a dual-reporter gene (such as mCherry/Neo). This allows cells transduced with the vector to be selected and/or visualized.
pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.