The PAM (Protospacer Adjacent Motif) is a short DNA sequence immediately following the DNA sequence targeted by the CRISPR-Cas9 system. It is essential for the Cas9 enzyme to recognize and cut the target DNA.
The PAM sequence is typically 2-6 base pairs long, and its specific sequence varies depending on the type of Cas protein used. For example, the PAM for the widely used SpCas9 (from Streptococcus pyogenes) is "NGG," where "N" can be any nucleotide.
The PAM sequence is crucial for target recognition by the CRISPR-Cas9 system. The Cas9 enzyme binds to the PAM sequence before unwinding the adjacent DNA to check for a match with the guide RNA.
For the commonly used SpCas9 from Streptococcus pyogenes, the PAM sequence is "NGG," where "N" can be any nucleotide.
The PAM is critical for Cas9 to bind to DNA. Without a PAM, Cas9 will not bind to the DNA sequence.
In bacteria, the absence of PAM sequences near CRISPR arrays prevents Cas9 from targeting the bacterium's own genome.
Different Cas proteins recognize different PAM sequences. For instance, SaCas9 from Staphylococcus aureus recognizes the "NNGRRT" PAM.
The requirement for a PAM sequence increases the specificity of CRISPR-Cas9, reducing the likelihood of off-target effects.
Scientists have engineered Cas9 variants that recognize different PAM sequences, expanding the range of possible DNA targets.
The presence and location of the PAM sequence affect the efficiency of CRISPR-Cas9 binding and cutting.
The specificity provided by the PAM sequence is used in gene editing, gene regulation, and CRISPR-based diagnostics.
In nature, the PAM sequence helps bacteria identify and cut foreign DNA, such as that from viruses, while protecting their own genome.
When designing CRISPR experiments, it is crucial to select target sites with the appropriate PAM sequence to ensure successful Cas9 binding and activity.
The PAM sequence is an evolutionary adaptation that allows bacteria to use CRISPR systems to combat viral infections effectively.
Beyond SpCas9, other Cas9 proteins, like Cpf1 (now called Cas12a), recognize different PAM sequences, further diversifying CRISPR applications
Cas9 first binds to the PAM sequence before unwinding the adjacent DNA to check for a complementary match with the guide RNA, initiating the cutting process only if the match is found.