Using gRNAs in the CRISPR/Cas9 system is a good reverse genetic approach to target a gene. However, a single gRNA target does not always result in a null. Here, we describe a method which will put 4 gRNAs all under the U6 promoters on a single molecule. When all 4 are targeted to a single gene, we consider this carpet bombing the gene. Not only is it extremely likely to cause a frame shift and null allele, but can also cause various sized deletions within the gene.
A graphical depiction of carpet bomb construction
The details for constructing a carpet bomb are contained here: Carpet Bomb tranposon construction protocol (Downloads an Excel Spreadsheet)
To design the primers referenced, consult here:CarpetBomb_PrimerDesign-v4 (Downloads an Excel Spreadsheet)
The sequence of the final plasmid (with N’s in place of the 4 gRNAs) is here: Generic_CarpetBomb_Transposon