How you raise the efficiency of transfection with CRISPR-Cas9 system (knock-outs of genes)?

12 October 2023 1 8K Report

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?

Robert Adolf Brinzer

Use two gRNAs to create larger deletions. Consider knocking in a cassette with a selection marker. If here is a big deletion you can detect the knockout by PCR.

Also transfection is the generic process of getting DNA into cells and independent of the efficacy of your CRISPR. To improve transfection consider having a DNA nuclear targeting sequence on your plasmid.

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