The PAM sequence is a critical determinant of where CRISPR systems can edit the genome. For the widely used Streptococcus pyogenes Cas9 (SpCas9), the PAM is NGG, meaning any nucleotide followed by two guanines must be present immediately downstream of the target site. This requirement exists because PAM recognition is the first step in target engagement: Cas9 scans DNA for PAM sequences and only unwinds and interrogates the adjacent sequence when a PAM is found, providing an initial layer of target discrimination.
The PAM requirement can limit the targetable sequence space, motivating extensive efforts to engineer Cas variants with relaxed or altered PAM preferences. Researchers at the Broad Institute and elsewhere have developed SpCas9 variants like xCas9 and SpCas9-NG with broadened PAM compatibility, and the near-PAMless SpRY variant can target almost any genomic sequence. Mammoth Biosciences and other companies prospecting novel CRISPR systems from diverse microbial sources have identified Cas proteins with distinct PAM requirements, expanding the toolkit for both research and therapeutic applications.
Understanding PAM biology is also important for predicting and minimizing off-target editing. Off-target sites must contain a PAM sequence to be cleaved, which partially constrains the universe of possible off-target locations. Computational off-target prediction tools incorporate PAM sequence requirements along with guide RNA homology to rank potential off-target sites. For therapeutic applications, PAM-dependent off-target profiling methods like GUIDE-seq and DISCOVER-Seq provide experimental validation of editing specificity, an essential component of the safety evaluation for any CRISPR-based therapy.