Question (2321)
Question
Which strand is the PAM site on? Do I need to reverse complement sequences for sgRNA cloning?
ABM community
Verified customer
Asked on Jan 16 2026
Answer
For SpCas9, the PAM sequence is NGG, and it is located on the non-target strand. The sgRNA binds the target strand (the strand complementary to the non-target strand sequence, excluding PAM).
As a general design rule:
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Identify the PAM (NGG) on the non-target strand
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The 20 nt protospacer immediately upstream of the PAM is the guide sequence used for sgRNA design
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Cas9 cuts approximately 3 bp upstream of the PAM
When cloning sgRNAs, reverse-complement requirements depend on the specific cloning system and oligo design format; customers should follow the cloning instructions for the backbone being used.
ABM Scientific Support
Answered on Jan 16 2026