Carpet bombs

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.

CarpetBomb Overview

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 plasmids used are here: U6-21_gRNA precursor transposon U6-9-Template_A U6-11-Template_B U6-6-Template_C

The sequence of the final plasmid (with N’s in place of the 4 gRNAs) is here: Generic_CarpetBomb_Transposon

 

 

Contributors:

(1) Stephen Johnson lab – Rob Tryon, Tom O’Reilly-Pol, and James Allen    (Washington University in St. Louis)
(2)  Ting Wang lab – Josh Jiang     (Washington University in St. Louis)
(3)  Wenbiao Chen lab – Y. Lin    (Vanderbilt University)

 

 

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