We have deposited two plasmids generated by drs. Sudeepta Kumar Panda and Sanjay Boddul, used in our recent PNAS paper (Panda et al, PNAS, 2020), to Addgene.
The first plasmid is a development of the LentiGuide-Puro from the Zhang lab where Sudeepta inserted a P2A-EGFP cassette enabling both Puro selection and EGFP based selection. We typically use the EGFP signal to evaluate the MOI and monitor the efficiency of puro based selection:
The second plasmid was developed from the LentiGuide-Puro-P2A-EGFP by Sanjay to also include an RFP reporter in the stuffer region driven by a bacterial promoter. This enables you to rapidly evaluate if bacteria expanded on agar/amp plates have taken up constructs where the stuffer is intact or not. This is very helpful to evaluate how well the cloning of a CRISPR/gRNA library, replacing the stuffer, into the plasmid has worked. A big thanks to the molecular biology wizard Erik Holmgren for help with the mRFP1 sequence.