Depending on your method of assessment, your editing efficiency may be underrepresented. Mismatch endonucleases like T7EI do not detect single base changes, and under-estimate actual editing compared to direct sequencing.
In addition, not every sequence associated with a PAM site performs the same. For example, polymorphisms in the protospacer binding site may reduce editing efficiency. Base mismatches also become more detrimental to editing the closer they are to the PAM site.
We recommend that you try 2 or 3 different PAM sites in your gene of interest to identify a site that provides optimal editing efficiency. Also, be sure to include control experiments. Alt-R™ CRISPR-Cas9 HPRT Positive Controls
and Alt-R™ CRISPR-Cas9 Negative Controls
are available for human, mouse, and rat.
Please feel free to contact us if you need additional assistance; having the results of your control experiments available will facilitate our ability to help you. firstname.lastname@example.org