Hi Nishant, this is indeed a bit puzzling, although sometimes indels are very large in NHEJ after Crispr/Cas9 double-stranded cuts, they should not occur in all clones, and your flanking region is large enough to account for most if not all indels...

Would it be possible that you have a problem with the DNA? maybe you extracted too little? Or the prep was not good enough for some reason? Did you have a look at your gDNAs on gel (on a 0.7% agarose gel you should see a clear band running very high, close to the wells... You might want to compare the wt DNA you use for control with your clones DNAs to see if they are equal amounts and equal integrity). Do you run a positive control PCR on your genomic DNAs, on a locus or gene that is not affected by your CRISPR/Cas9 approach? Do you have any evidence that after subcloning the cells were growing enough (your deletion might affect cell survival or division or growth...)?

Good luck!

Hi Pietro, Indeed it is puzzling . 

I have high DNA  concentration (400ng/ul, A260/A280 1.9) and quality is good.

I haven't tried gDNA on gel but i did PCR for WT cells and sent the product for sequencing.Sequence of WT is perfect. I can check gDNA of WT and clones on gel.

I always run WT as positive control because WT is non transfected cells.I always get the pcr band for different set of primers and 100% sequence matches.

All KO clones photogenically behave the same .When compare to WT they behave differently.which is good  for me. 

Thanks.

Are you sure you have the same amount and quality of DNA?  Sometimes in this very situation I've seen the PCRs collapse, because it's often easier to get large amounts of high quality wild type DNA, than it is to get DNA from all the clones. 

Hi John, i am sure DNA is good amount of good quality DNA.

Hi

Currently I have the same problem. Did you find the reason why this happens?

thank you

Hi Barbara! I have the same problem too. Did you risolve this?

thank you!

Unfortunately I have the reached the same wall (in plant though). Anybody have a solution yet? I am planning to go forward with protein and enzyme assays as it was a protein knockout. Any thoughts?

Hi everybody!

In my case, the plasmids with the gRNA were the problem itself, they didn't work well.

Good luck!

I found answer for my problem. In fact I learned that it is not a problem. I was targeting tandem genes, so obviously I had targeted different genes stringed together using a single common sgRNA and thereby making a double double nick, which removed the whole piece out of my DNA (almost 10kb - not yet confirmed - work is under way). As I was trying to knockout these genes, good for me, though I did not think about such a consequence in the beginning.