The CRISPR gene-editing system has transformed biological research, offering a precise way to modify DNA. While guide RNA directs the system to specific DNA sequences, the Protospacer Adjacent Motif (PAM) sequence is equally important for CRISPR to function.
What is the PAM Sequence?
The PAM sequence is a short, specific DNA sequence, typically 2-6 base pairs in length, found immediately next to the target DNA sequence that the CRISPR-Cas system aims to modify. The Cas enzyme, commonly Cas9, actively seeks out this sequence. Without this precise sequence, the Cas enzyme cannot attach to the DNA or perform its cutting action. The PAM sequence is a component of the invading viral or plasmid DNA, but it is not found in the bacterial host’s own genome, which helps the system distinguish between self and non-self DNA.
For instance, the most widely used Cas9 enzyme, derived from Streptococcus pyogenes (SpCas9), recognizes a specific PAM sequence of 5′-NGG-3′, where “N” can be any nucleotide base followed by two guanine (“G”) nucleotides. The PAM is not part of the guide RNA, but its presence on the target DNA is necessary for the CRISPR-Cas system to initiate activity.
How PAM Guides Gene Editing
The PAM sequence plays a direct role in guiding the gene-editing process. The Cas enzyme, such as Cas9, first scans the DNA for a recognizable PAM sequence. This initial recognition is a prerequisite; the enzyme will not proceed without finding the correct PAM. Once a suitable PAM is identified, it signals the Cas enzyme to begin unwinding the adjacent DNA double helix.
This unwinding allows the guide RNA (gRNA) to then check for a complementary match with the target DNA sequence, also known as the protospacer. The PAM acts as an anchor point, facilitating the initial binding of the Cas protein to the DNA and enabling the subsequent pairing between the gRNA and the target DNA. If the guide RNA finds a perfect or near-perfect match, the Cas enzyme then proceeds to cleave both strands of the DNA, typically 3-4 nucleotides upstream from the PAM sequence.
PAM’s Role in Specificity and Versatility
The specific requirement for a PAM sequence is a key aspect of CRISPR’s precision, ensuring that the Cas enzyme only cuts at the intended genomic location. This mechanism effectively minimizes the chance of off-target edits, which are unintended modifications at other sites in the genome. The Cas enzyme will not bind to or cleave a DNA sequence if the appropriate PAM is absent, even if the guide RNA matches a different DNA sequence elsewhere. This specific requirement serves as a safeguard, enhancing the accuracy of gene editing.
The diversity of PAM sequences recognized by different CRISPR systems contributes greatly to the versatility of the technology. Various Cas enzymes, such as Cas9 and Cas12a, originate from different bacterial species and recognize distinct PAM sequences. For example, while SpCas9 recognizes the 5′-NGG-3′ PAM, Cas12a nucleases, like those from Acidaminococcus sp. (AsCas12a), typically recognize thymidine-rich PAMs such as 5′-TTTV-3′, where “V” can be A, C, or G. This variety in PAM recognition expands the range of genomic locations that can be targeted for editing, offering scientists more options when designing gene-editing experiments.
Understanding and even engineering PAM sequences can broaden the applicability of CRISPR tools for gene therapy and research. Scientists have developed engineered Cas9 variants with altered PAM specificities, allowing them to target sequences not accessible by the original Cas9 enzyme. For instance, some engineered Cas12a variants can recognize a broader range of PAMs, including 5′-TTTN-3′, where “N” is any nucleotide, further increasing targeting flexibility. These advancements provide greater flexibility in selecting target sites, which is especially beneficial for complex genome editing projects like gene knock-outs or knock-ins where specific genomic locations must be precisely modified.