I am working on HEK293T cells and trying to make gene specific knockout cell line by using Crispr Cas9, but at the end of single clones the success rate is very low.
The efficiency is dependent on many different factors. What are your exact conditions? Which transfection method? Which CRISPR delivery system? How many gRNAs? Were the gRNAs tested before concerning efficiency (eg by simple TIDE analysis)? Where (in the gene) are the gRNAs located? Did you analyze the editing efficiency in the transfected bulk culture?
Before starting a knockout experiment, I usually test 2-5 gRNAs and chose the best ones. For KOs, the gRNAs should ideally be located around the transcription start side or within the first coding exons. In my hands, I can achieve the highest knockout efficiencies with the simultaneous use of 2-3 guides that are located about 50 bp apart from each other leading to frameshift deletions. If possible, you should use recombinant Cas9 protein pre-assembled with synthetic modified sgRNAs (=Cas9-RNP) rather than plasmid- or mRNA-based approaches. In my opinion, electroporation-based delivery of the components works the best, Lipofection, PEI etc. does not work so well for the RNP delivery. However, it is also possible, that you hit a locus that is hard to edit and that you cannot find any “active gRNA”, but active genes should usually work.
I hope this helps,
Floriane
hi, i think you should go for RNP mediated and use nuclear localization signaling. there are several tactics that can minimize cytotoxicity, in my opinion vector mediated delivery can only be used when your cell are nicely compatible to plasmid. Mechanical method may damage the cell...on other hand you should choose wisely the sgRNA, along with it we do an in vitro cleavage....are you getting success in it , if you are succeeding in IVC, then there is good chance that you can succeed. yeah, sure you can go for multiple guide RNAs.
hope this answer satisfies you...i am not a pro. thanks
there are various techniques to properly knock out the genes, such as:
1. targeting the first exons of a gene.
2. knocking our one or two nucleotides (not three)
3. targeting an active-site, if your gene is translated to protein later. it is a key to successfully inactivating the function.
3. using anti-CRISPR protein to reduce the number of targeting.
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