Did u take a similar cell number in both control and treated conditions?. because in 18s the amount of amplification looks slightly less. Though not a prominent difference.

Does your gRNA targets any of the UTR? say if it's in 5'UTR the region might have had some repressor binding elements that once after editing by gRNA would have caused an upregulation in expression. If the gRNA had targeted 3'UTR that might have had mRNA stability associated elements the transcripts of the gene would be sticking around for a longer time than in control.

Vasanth Thamodaran

Hi! yes I took the same amount of cdna for pcr in all the conditions.

As far as gRNA is concerned, it has been designed to target exon 2 of my gene. It is not targeting any UTR.

Thankyou

Hi Nisha,

In my opinion the apparent upregulation of your target gene is unrelated to Cas9 activity. The indels production, however, can be easily validated by sanger sequencing PCR and TIDE to clear all doubts. KO production generally leads to nonsense mediated RNA decay, so you should eventually observe the opposite.

I would recommend you to check your genomic DNA for indels production. The explanation of Vasanth is valid up to a point. Since you are dealing with an heterogeneous population I don't expect 1) all the cells to be edited 2) all the edited cells to have the same mutation.

After genomic DNA analysis you can have a better idea and:

If you do not have any indels

1) Check the integrity of your lentiviral backbone

2) Check the integrity of the U6 sgRNA expression cassette by sanger sequencing

-If everything is ok, then your sgRNA is not working. Design and clone a different one for another round of experiments

-If lentivector or hU6 cassette have mutations/rearrangement repeat the cloning using Stbl2 and proceed only after restriction digestion and Sanger sequencing of the cloned sgRNA

If you have indels

The upregulation effect could be due to:

1) Differences in transcriptome between Puro treated and untreated samples

-Allow transduced cells to grow in absence of Puro for 1 week before repeating RNA extraction

-Add an internal housekeeping gene for checking RNA and cDNA integrity

2) Is your qPCR amplicon around the sgRNA site? If not include one

If you have indels but the RNA is still upregulated after all these controls, it means your protein is still produced but with a different isoform (Exon 2 is probably skipped or protein production is resumed from the first available ATG after your edit). It happened to me in a recent publication (Article Canonical Wnt Pathway Controls mESC Self-Renewal Through Inh...

Sorry for the long answer but I wanted to be exhaustive. If I were you, I would not focus too much on the RNA expression levels however, but rather validate the sgRNA efficiency first by standard indels determination methods. Once you have a positive hit you move forward with RNA and protein analysis.

Hope it helps

Francesco

Hi Francesco Aulicino,

Thankyou for your answer. It would be of great help and it has definitely given me direction to move further with my experiments.

Thankyou so much!!

Nisha