Three new plasmids that were made during our work on lncRNA DIGIT are now available through Addgene. Here's the brief description and what you can do with them:
1) pAAVS1-shRNA (Plasmid #82697)
This plasmid was made based on PLKO shRNA backbone. You can design and clone your shRNA of interest just like you would for PLKO constructs. In brief, you would want to synthesize two annealing oligos according to GPP portal guides. For cloning, you would want to digest pAAVs1-shRNA plasmid by EcoRI and AgeI, and then clone the annealed oligos.
What's different about this plasmid is that your shRNA of interest will be flanked by two homology arms that can target the construct to the AAVS1 locus. This will make one able to efficiently make stable cell lines without using a virus. Of course, you would want to co-transfect the plasmid with a CRISPR/gRNA or TALEN construct to make a double-strand break in the AAVS1 locus. These constructs are already available on Addgene (this one should work). The construct constitutively expresses puromycin resistant gene for selection.
2) pgRGFP (Plasmid #82695)
This is a backbone that expresses the gRNA of interest and it also constitutively expresses GFP. This plasmid is made based on PX330, and cloning gRNAs follows the same principle (BpiI digestion of the backbone and cloning of annealed oligos).
3) pCRE-iRFP670 (Plasmid #82696)
This plasmid constitutively expresses CRE recombinase and iRFP670 for cell sorting. It's a great plasmid for genome-editing situations where you want to flox out an insert flanked by lox-p sites. The iRFP670 is bright far-red bright fluorophore that is easily detectable on FACS or cell sorter in APC channel.