Hi Sumit,

I achieved successful CRISPR KO of my gene of interest in various cancer cell lines using the general protocol below.

1) Design 2-3 guide sequences for your gene of interest using http://crispor.tefor.net/

2) Align your guide sequences against reference genome using Ensembl BLAST to make sure they recognize your gene of interest with minimal number of off-target alignments

3) Clone guide sequences into the pCas-Guide-EF1a-GFP CRISPR vector (Origene GE100018)

This company has an amazing CRISPR manual- https://cdn.origene.com/assets/documents/crispr-cas9/crispr_manual.pdf

4) Transfect your target cells in the 96-well format and confirm GFP expression after 48-72 hours

5) 72-96 hours following transfection, get the cells flow sorted based on GFP expression and then get the GFP-expressing cells single-cell sorted into 96-well plates

6) Some cell lines cannot form single-cell colonies. Hopefully your cells can- it should take a couple weeks for decently-sized colonies to form.

7) When you have enough cells for each of your colonies, check for decreased protein expression of your gene of interest by Western blot and then perform Sanger sequencing to validate gene editing. CRISPR KO efficiency isn't that high- only 10% of all my single-cell colonies exhibited decreased protein expression.

Hope this helps.

Best,

Nesli

Muhammad Naeem

Thank you Deniz Nesli Dolcen