Hi, I´m currently planning to knock-out a single gene in human cell line. I have basic idea of how CRISPR works, but I have no previous experience with generating knock-out cell lines, so I want to make sure the design is good and also possibly time and work efficient. So my question is: what markers do you use, what is your method of choice for selecting single clones, what is your favourite way of genotyping?
So far what I heard, it is better to use stop codon knock-in than to rely on random mutations, do you agree? Should I knock in some antibiotic resistance as well?
I was looking for these informations on the web, but I wasn´t very succesful. So I think your replys can make up a nice summary in the end.
Thank you very much for your help.
JS
Hi Jakub,
To this very broad question you could get a very long answer to but, for starters, I'll try to make it short:
- Read some kind of CRIPSR tutorial, as addgene's CRISPR 101 ebook, or something similar, I'm sure there are dozens out there. Maybe some nature (methods) papers wouldn't hurt.
- In general "normal" knock outs via NHEJ are easier to obtain than knock-ins, so I'd start with that.
- choose an as specific as possible guide, e.g. using crispor.org, in an early exon - preferably exon 1.
. Decide on the actual cas9-guide format and delivery route (Plasmid, virus, rec. prot., (IVT)RNA, electroporation, lipofection etc.)
- If you do single cells (serial dilution or 0.5 - 1 cell/well in 96wells) you can do sanger sequencing. If you use the bulk you have to use some NGS method.
- If antibody available stain for expression of your protein.
Good luck,
Thomas
Hi Jakub,
I think you could use this article to guide you to the right direction (among others). Article Genome engineering using the CRISPR-Cas9 system
Hi Jakub,
If I were you, I think CRISPR-Cas9-mediated knockout is much more reasonable. You can use a lentivirus-mediated system, and infect your cells. You can either select single clone using serial dilution or FACS sorting, or just select the positive cells using puromycin treatment. There is also lentivirus plasmid with GFP tag available in some companies, such as OpenBiosystem.
You can simply detect knockout efficiency using immunoblots, or you can do sequencing as well. It depends on your project budget.
Hope this information is helpful for you!
Jennifer
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