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.
