Hello! Can you give me some tips how to do CRISPR-Cas9 more effectively? Maybe you can give me some advice on this topic. This technology is new for me, I tried to conduct the experiments to knock-out genes, but they weren't successful. How do you detect knock-outs? What is the best combination of conditions to carry out CRISPR-Cas9?
You usually detect knock outs by knocking in a cassette with a selectable marker gene or cutting in two locations which causes a deletion large enough to be detected with PCR. Double check that your plasmids are using promoters appropriate for your organism. If you are simultaneously trying to knock in a marker then you can combine the system with single strand annealing proteins used for recombineering (lambda red for prokaryotes and ICP8 for eukaryotes).