CRISPR-Cas9 PAM
BioInnovatise CRISPR Team
Updated March 24, 2025
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CRISPR-Cas9 PAM (Protospacer Adjacent Motif) is a specific DNA sequence that’s essential for the CRISPR-Cas9 gene editing system to function properly.
The PAM is a short DNA sequence (typically 2-6 base pairs) that must be present immediately following the target DNA sequence for the Cas9 enzyme to recognize and cut the DNA.
For the most commonly used Cas9 enzyme from Streptococcus pyogenes (SpCas9), the PAM sequence is 5′-NGG-3′, where “N” can be any nucleotide followed by two guanine (G) nucleotides.
CRISPR-Cas9 PAM Core Functions
The CRISPR PAM services several critical functions including:
- It helps the Cas9 enzyme distinguish between self and non-self DNA.
- It acts as a binding site for the Cas9 protein.
- It triggers the unwinding of the target DNA, allowing the guide RNA to form a complex with the target sequence.
- It positions the Cas9 nuclease domains to cleave the DNA at a predetermined precise location
The PAM requirement is also a limitation of CRISPR gene editing because it restricts where the Cas9 can make its cuts in the genome. Some scientists have been able to work around this by custom engineering Cas9 protein variants with different PAM requirements, using Cas proteins from other bacterial species, or adopting new CRISPR gene editing techniques.
CRISPR-Cas9 PAM's Role During CIRPSR Knock-In or Knock-Out Projects
The PAM is absolutely integral during CRISPR knock-out and knock-in productions. However the goals of each gene editing project will use the PAM sequence in different ways.
Note: PAM availability can sometimes limit where you can make precise cuts, which can be challenging if you need to target a very specific location for your knock-out or knock-in.
CRISPR Knock-out:
- The PAM sequence must be present near the target site for Cas9 to create a double-strand break.
- When designing a knockout strategy, researchers must identify target sequences with appropriate PAMs.
- Target early exons or critical domains where the PAM is available.
After Cas9 cuts at the PAM-adjacent site, the cell’s error-prone repair mechanisms (NHEJ) often introduce insertions or deletions that may disrupt the gene function.
CRISPR Knock-In:
- Design the gRNA to cut near the location for a new sequence.
- The provided donor DNA template (containing your desired insertion) doesn’t need to contain the PAM.
- Our CRISPR team designs donor templates to match the desired gene edit requirements.
- However, many strategies intentionally modify the PAM sequence in the donor DNA to prevent Cas9 from cutting the newly inserted sequence after integration.
- This approach ensures the knock-in stays intact after successful cell line integration.
If you have a question about CRISPR PAM for your CRISPR knock-out or knock-in project, contact our CRISPR team.
Our CRISPR team is excited to bring your project to life. Learn more about our quick turnaround CRISPR-Cas9 services. Our team offers a CRISPR library and produces custom CRISPR vectors, sgRNA cloning service, cassette and donor vector construction design services, and CRISPR knock-out and CRISPR knock-in services.
Want to learn more about the latest in CRISPR genome-editing based research? Our colleagues at ScienceDirect and Genetic Engineering & Biotechnology News continuously collect and publish the latest information on CRISPR based research.
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