CRISPR technology has transformed the field of gene editing, offering a precise method for modifying genetic material. Within this system, a specific DNA sequence known as the Protospacer Adjacent Motif (PAM) plays a foundational role in enabling its function. Understanding the PAM is important for comprehending how CRISPR accurately targets and edits genes.
What is PAM?
PAM stands for Protospacer Adjacent Motif, a short and specific DNA sequence typically ranging from 2 to 6 base pairs in length. This sequence is located immediately next to the target DNA sequence that the CRISPR-Cas system is designed to recognize and cleave. It is important to note that the PAM is found on the invading viral or plasmid DNA, not within the bacterial host’s own CRISPR array or the guide RNA that directs the Cas enzyme.
For instance, the commonly used Cas9 enzyme from Streptococcus pyogenes (SpCas9) recognizes a canonical PAM sequence of 5′-NGG-3′, where “N” represents any nucleotide base, followed by two guanine (G) bases. Different Cas enzymes, such as Cas12a, recognize distinct PAM sequences, like 5′-TTTV-3′ (where V can be A, C, or G). The presence of this specific motif acts as a marker on the DNA.
PAM’s Role in Target Recognition
The PAM sequence enables CRISPR-Cas system activity. The Cas enzyme, such as Cas9, first identifies and binds to the PAM sequence on the target DNA. This initial binding event is a prerequisite for the Cas enzyme to proceed with checking for complementarity between its guide RNA and the adjacent target DNA sequence. Without the correct PAM, the Cas enzyme cannot effectively bind to the DNA, preventing the gene-editing process from beginning.
The PAM also distinguishes between the bacterial host’s own DNA and foreign genetic material, preventing self-targeting. The bacterial CRISPR array, which stores segments of foreign DNA, intentionally lacks the PAM sequence adjacent to its “spacers” (the stored foreign DNA fragments). This design ensures that the Cas enzyme does not mistakenly cut the bacterium’s own genome, maintaining genomic integrity.
How PAM Guides Gene Editing
The PAM facilitates the gene-editing process through a precise, multi-step mechanism. The Cas enzyme begins by scanning the DNA for its specific PAM sequence. Upon recognizing and binding to the PAM, the Cas enzyme induces a localized unwinding of the DNA double helix. This unwound region allows the guide RNA to access the target DNA sequence, which is positioned next to the PAM.
If there is sufficient complementarity between the guide RNA and the target DNA sequence, the Cas enzyme undergoes a conformational change. This change enables the enzyme to precisely cleave both strands of the DNA.
The Impact of PAM on CRISPR’s Versatility
The specific PAM sequence required by different Cas enzymes significantly influences the range of genomic locations that can be targeted for editing. While the commonly used Streptococcus pyogenes Cas9 (SpCas9) requires a relatively frequent NGG PAM, other Cas enzymes recognize different PAM sequences. For example, Cas12a enzymes often recognize T-rich PAMs like TTTV. This diversity in PAM requirements expands the overall targeting flexibility of CRISPR technology, allowing scientists to access and edit a broader array of genes or specific sites.
Conversely, the requirement for a specific PAM also presents a limitation for gene editing. Not every desired editing site in a genome will have a suitable PAM sequence nearby, which can restrict targeting options. To overcome this, researchers have engineered Cas9 variants with altered PAM specificities or explored other Cas nucleases with different PAM preferences, such as “near-PAMless” Cas9 variants.