I have a small challenge with a reporter assay we use to determine sgRNA spacer efficacy. The reporter vector does not express GFP, or at a very low level, after incorporation of my spacers. I had this before. We solved that partly by dividing the spacers in two parts and ligate them in separate vectors. One vector had GFP expression after the split; the other one had no GFP expression. But both cultures were Puro resistance which is present on the reporter vector.

In the meanwhile I developed more sgRNAs and needed a new reporter to validate these sgRNAs. Because I still had the spacers from previous reporter, I decided to combine my new spacers and the ones left over from the previous reporter.  Again this reporter has almost no GFP expression.

How are the reporters made:

The spacer sequences are linked behind each other and a positive control is added. Restriction sites are added with AgeI at the 5’ end and MluI at the 3’ end. The sequence is translated into an amino acid sequence. The sequence must be in-frame between the two restriction sites. Possible stop codons are removed. The sequence is then order as a gBlock and ligated into the vector. Colonies are checked with restriction enzymes and positive colonies are sequenced.  

For this new reporter I made sure that the spacers from the previous reporter were spread through the sequence, creating a different amino acid sequence than the previous reporter. The reporter is sequenced and all seems fine (in-frame and no mutations). Reporters with 40 spacers were made in our lab. I have now 21 spacers. Apparently 1 in 5 reporter vectors don’t express GFP.

Are there any ideas about the cause of this GFP inhibition? (If you can call it like that). Is it caused by some amino acid? Or ….