Top Questions

My CRISPR-Cas9 genome editing efficiency seems very low. How can I improve my experiments?

Possible reasons for low editing efficiency

  • 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.
  • 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.

Recommendations

  • Try 2 or 3 different PAM sites in your gene of interest to identify a site that provides optimal editing efficiency.
  • Include control experiments to help monitor or optimize CRISPR reagent delivery. Alt-R® CRISPR-Cas9 HPRT Positive Control cRNAs and Alt-R CRISPR-Cas9 Negative Control cRNAs are available for human, mouse, and rat.
  • If you are using electroporation methods, including Alt-R Cas9 Electroporation Enhancer could improve transfection efficiency and, therefore, editing efficiency.
  • Please contact us at applicationsupport@idtdna.com for additional assistance; having the results of your control experiments available will facilitate our ability to help you.
Tags:
  • Cas9
  • CRISPR
  • Alt-R
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