How to ensure accurate gene editing in cell lines

[brightpairs56]

I'm new to CRISPR knock-in experiments, I'm curious about the stages involved in your lab's quality control process. What are the steps you take to verify the accuracy of the gene editing in your cell lines? Any insights would be greatly appreciated. Thank you.

 

[nucLe01]

Nice, welcome to the world of CRISPR!
I can't disclose much but for knock-ins, our QC usually starts with PCR to confirm the insertion.
Then we move to Sanger sequencing of the edited locus to check for precise integration and detect any indels.
For more critical projects, we often do ddPCR for copy number verification and sometimes even NGS for off-target analysis.

 

[Crispy Fry]

I'm new to CRISPR knock-in experiments, I'm curious about the stages involved in your lab's quality control process. What are the steps you take to verify the accuracy of the gene editing in your cell lines? Any insights would be greatly appreciated. Thank you.

Welcome to the gene-editing endeavor. Usually, we start with PCR and Sanger to confirm the insert and then run some sequencing to make sure everything's where it is supposed to be. Don't forget to double check before trusting the cells to behave! Crazy times.

 

[Angelina.Mc]

Welcome to the world of CRISPR knock-ins, definitely a lot to juggle at first. In our setup, after transfection, we usually start with PCR screening to check for integration at the right locus, followed by sanger sequencing to confirm the precise edit and if that looks good, we isolate clones and do a second round of sequencing