Hello experts in the CRISPR/Cas9 field,
I am new at CRISPR/Cas9 field, I tried to perform CRISPR/Cas9 KO HEK293 cells line but it failed.
I transfected CRISPR/Cas9-gRNA into HEK293T cells via lipofectamine 3000, after 48 hours, I harvested the transfected-cells, extracted genomic DNA, amplified the target region and performed SURVEYOR assay, unforturnately, I can't see any cleavage.
I am worried about whether either the transfection efficiency is low???
Is there any good suggestion how it happens and what I should do to improve it?
How can I increase CRISPR/Cas9 targeting efficiency?
Thank you very much in advance for your time
With my best regards!
Hi Rich, Hek293T is standard cell line for work with CRISPR and gives high transfection efficiency with lipofectamins. So you should be more concerned about design of your sgRNA, quality of reagents etc. To eliminate concern about Tx efficiency a standard approach is to use GFP expression vector in the same experiment, visually check rough % for Tx (that could be your control gDNA sample too). One simple rule about sgRNA is that GC content of your sgRNA should be in 40-60% range, other % works too but that % GC gives higher success rate and % of indels for sgRNAs. Another useful approach is to design multiple sgRNA for your target and test a bunch of them, particularly if KO is your goal (for HDR too, but using just 100 bp window around target site) . You could use a validated sgRNA to the gene of your interest or any other gene for that matter, which should gives you around 50% or more indels rate, to make sure that your whole procedure works from the start to the end.
Hi Rich, I agree with Dmitry. An empty-vector control with a tag is a must-do, and you can evaluate transfection efficiency. Another question is what's the size of plasmid DNA? It is usually difficult to transient transfect a huge plasmid. You can try a lentivirus-mediated system to infect cells, which may work much better than lipo-system.
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