After CRISPR editing, what verification methods confirm whether an edit occurred?

• A. Western blot of all proteins
• B. qPCR of housekeeping genes only
• C. Flow cytometry of cell surface markers only
• D. PCR amplification and sequencing of the target locus, plus off-target analysis if needed

Answer: (D)

Verifying a CRISPR edit hinges on confirming the exact genetic change at the intended site and checking for any unintended edits. PCR amplification of the target locus followed by sequencing shows precisely what alteration occurred—whether the desired nucleotide change, insertion, or deletion took place—and reveals the edited allele’s sequence and, if needed, its zygosity in the cells. If the editing approach raises concerns about off-target activity, off-target analysis examines other potential sites for edits, using targeted sequencing of predicted sites or broader methods, to ensure edits are specific.

Protein-level or expression-level tests don’t confirm the genome edit itself: a Western blot of many proteins wouldn’t prove a specific nucleotide change, qPCR of housekeeping genes only reports RNA levels, and flow cytometry of cell surface markers reflects phenotypic outcomes rather than the exact DNA modification.