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To use the PE3b or PE5b systems, a PAM needs to be present on the non-edited strand close to the edit. A nicking sgRNA can then be designed such that it can only bind and direct nicking of the non-edited strand after reverse transcription and flap equilibration have occurred. Such a PE3b/PE5b nicking sgRNA has a spacer that is perfectly complementary to the edited DNA sequence, but contains mismatches with the unedited sequence.
