The CRISPR-Cas9 system provides a powerful tool for precision genome editing, allowing scientists to make highly specific changes to a cell’s DNA sequence. Targeted editing relies on a molecular checkpoint that ensures the Cas9 enzyme cuts at the correct location. This checkpoint is the Protospacer Adjacent Motif (PAM), a short DNA sequence that acts as the necessary signal for the enzyme to begin its work. The specific PAM sequence differs depending on the source of the Cas9 enzyme, dictating where a particular Cas9 variant can operate in the genome.
Defining the Protospacer Adjacent Motif (PAM)
The Protospacer Adjacent Motif (PAM) is a short, conserved sequence of DNA found immediately next to the target site. This sequence is not part of the genetic material that the guide RNA (gRNA) is designed to match, but its presence is required for the Cas9 enzyme to function. In the natural bacterial immune system, the PAM serves to distinguish the cell’s own DNA from foreign invaders, such as viruses.
The Cas9 enzyme will only cut DNA that contains the specific PAM sequence, which is present on the invading viral DNA but absent from the host bacteria’s own CRISPR locus. This requirement prevents the immune system from mistakenly targeting and destroying its own genome. Therefore, the PAM acts as a “self” versus “non-self” recognition signal.
In genome editing applications, the PAM sequence determines where the Cas9-gRNA complex can bind and cleave the DNA. Different Cas9 enzymes (orthologs) recognize distinct PAM sequences, which is a major factor in selecting the appropriate Cas9 for an editing task. Without the correct PAM sequence positioned next to the desired target sequence, the Cas9 enzyme will ignore the site, regardless of how well the guide RNA matches the target DNA.
Identifying the SaCas9 PAM Sequence
The SaCas9 enzyme is derived from the bacterium Staphylococcus aureus and is an important tool in gene editing. This specific enzyme requires a Protospacer Adjacent Motif (PAM) sequence that is longer than the one used by the common Cas9 variant, SpCas9. The specific PAM sequence required for SaCas9 activity is NNGRRT.
The letters in the NNGRRT sequence represent specific nucleotides and nucleotide groups.
Decoding NNGRRT
- The ‘N’ represents any of the four DNA nucleotides (Adenine, Cytosine, Guanine, or Thymine), meaning the first two positions are flexible.
- The ‘G’ stands for Guanine, a fixed requirement in the third position.
- The ‘R’ denotes a purine base (Adenine or Guanine), constraining the fourth and fifth positions.
- The ‘T’ represents Thymine, a fixed requirement in the sixth position.
While the standard SpCas9 enzyme recognizes the shorter NGG PAM, the longer NNGRRT sequence makes SaCas9 more restrictive in the number of sites it can target. However, the primary advantage of SaCas9 lies in its physical size; at only 1053 amino acids, it is significantly smaller than the SpCas9 enzyme. This reduced size makes SaCas9 easier to package into delivery vehicles, such as the adeno-associated virus (AAV). The ability to fit the entire editing system into a single AAV vector makes SaCas9 a valuable option for in vivo applications.
How the PAM Sequence Directs Genome Editing
The PAM sequence functions as the initial binding handle and activation trigger for the Cas9 enzyme. The Cas9 protein, guided by its single guide RNA (gRNA), scans the double-stranded DNA for a potential target site. Cas9 does not attempt to match the gRNA to the target DNA until it first successfully encounters the correct PAM sequence, such as NNGRRT for SaCas9.
Upon recognizing and binding to this motif, the Cas9 undergoes a structural change. This change destabilizes the adjacent double-stranded DNA, causing the DNA strands to locally unwind or “melt” near the PAM. The unwound DNA then allows the gRNA to test for complementarity by attempting to form base pairs with the target strand.
If the gRNA successfully hybridizes with the target DNA, a structure known as an R-loop is formed, confirming the correct target site. This successful binding and R-loop formation fully activates the two nuclease domains within the Cas9 protein, HNH and RuvC. Once activated, these domains cleave both strands of the double helix at a specific location, typically three base pairs upstream of the PAM sequence, creating the double-strand break necessary to initiate genome editing.