TITLE: Polycistronic plasmids for mutating genes
CASE NUMBER: C13607
ABSTRACT
The invention is a series of plasmids. Each plasmid drives the expression of (a) two (2) PolIII transcripts encoding CRISPR-based guide RNAs (gRNAs) that target a specific gene and (b) one (1) PolI transcript that expresses a polycistronic ORF encoding (i) Sp Cas9 protein fused to three (3) copies of the SV40 nuclear localization signal, (ii) EGFP, (iii) Herpes simplex virus TK, and (iv) puro. This long, four (4) protein-encoding ORF is flanked by loxP sites so that it can be excised from the cell genome via expression of Cre recombinase.
FEATURES
1. The plasmid makes use of a Cas9-3xNLS fusion to obtain better nuclear localization of the Cas9 protein
2. The plasmid expresses Cas9-3xNLS as part of a polycistronic mRNA that also expresses (i) an FP for fluorescence detection/slection, (ii) HSV-TK for negative selection in the presence of ganciclovir, and (iii) puro, for positive selection of transfected cells.
3. The polycistronic ORF is flanked by loxP sites, which allow for its elimination upon expression of Cre recombinase in the cells, and subsequent selection of such deletion events by growing the cells in ganciclovir.
4. The puro gene is used as selectable marker, which selects for high-level expression of the polycistronic ORF over a short (3-8 day) period, generating considerable enrighment of mutated cells. In this regard, puro works far better than bsd, which is the other selectable marker option for a fast-killing antibiotic, and which does not result in any enrichment of KO cells.
5. The use of the 7sk promoter to drive expression of one of the gRNAS
6. The tandem use of the 7sk promoter together with the H1 promoter to target two sites in a single gene.
DECRIPTION DETAIL
The plasmid series allows any user to create mutations at specific sites within the genome, by (a) creating derivative plasmids that express gRNAs specific to a particular gene, (b) transfecting the derivative plasmid into a cell line or organism of choice, (c) selecting for puromycin-resistant cells/organisms, (d) expressing Cre recombinase, (e) selecting for ganciclovir-resistant cells, and (f) screening for cell lines or organisms that have mutations in the desired gene.
ASSOCIATED PUBLICATIONS: None at this time
