Protospacer Adjacent Motif or PAM (Part 8- CRISPR in Gene Editing and Beyond)
Welcome to the 8th part of the 40-part series on applications of CRISPR in Gene Editing and Beyond.
We have already discussed in Part 4 that the CRISPR systems in bacteria were evolved to defend bacteria against viruses. After the integration of protospacers into the CRISPR locus, the CRISPR locus is transcribed to form precursor-crRNA. After maturation, crRNA forms a complex with Cas proteins to create a search complex. And if the sequence of crRNA matches with the sequence of the DNA of the invading virus, then the Cas protein cuts the viral DNA (discussed in Part 6). Thus, cleavage of viral DNA destroys the invading viral genome and therefore protects the bacteria from the viral infection. But the viral DNA targeted by the search complex is the same sequence as the protospacer DNA in the CRISPR array. Because protospacers are the excised segments of the invading viral DNA.
So how exactly is Cas protein able to distinguish between itself and the enemy?
This is where PAM comes in. The PAM stands for Protospacer Adjacent Motif. PAM is a specific sequence of nucleotides, around 2–6 bp, that follows the protospacer sequence in a viral genome.
It is imperative to understand that PAM is a component of the invading virus or plasmid but is not a component of the bacterial CRISPR locus.
In the interference step of CRISPR system-mediated defense, specific Cas proteins recognize and bind the PAM sequence. The PAM recognition is necessary to facilitate the double-stranded DNA target’s unwinding, thereby triggering the base pairing between the crRNA and the DNA target followed by cleavage with the Cas proteins.
On the contrary, Cas nucleases cannot successfully bind to or cleave the target DNA sequence if the PAM sequence does not follow it. For instance, in Type II CRISPR-Cas systems, Cas9 recognizes the PAM sequence -NGG- when read in the 5’-3' direction’ where N is any nucleotide followed by 2 Guanine residues. This PAM sequence must be present for the Cas9 protein to know that it’s ok to latch onto and cut the viral DNA. On the other hand, the spacer sequences within the CRISPR array are not followed by “GG.” It means that the Cas9 cannot bind to the CRISPR array and thereby avoids cutting the bacterium’s own genome.
The PAM sequence “NGG ‘’ is associated with the Cas9 nuclease of Streptococcus pyogenes, whereas different PAM sequences are associated with the Cas9 proteins of different bacteria. For Instance, Staphylococcus aureus Cas9 recognizes “NNGRRT” PAM sequence where R is any purine may be A or G nucleotide. And Campylobacter jejuni Cas9 recognizes NNNVRYAC PAM sequence where V is A, G, or C nucleotide; and Y is T or C nucleotide.
We have already discussed in Part 7 that the type-1 CRISPR-Cas system utilizes the Cascade complex in the interference step and recruits Cas 3 protein to degrade viral DNA. In E. coli, the Cascade complex consists of a mature crRNA and five Cas proteins, Cse1, Cse2, Cas7, Cas5e, and Cas6e subunits. During the Interference step, the Cse1 subunit of the Cascade complex recognizes the PAM region of the viral DNA. It then positions the Cas3 protein adjacent to the PAM to ensure the cleavage of viral DNA.
Both Type I and II CRISPR-Cas systems search for foreign genomes during the interference step, requiring the presence of a PAM sequence and perfect protospacer-crRNA complementarity in the so-called seed region, which is located adjacent to the PAM. Thus the seed region can be defined as the region of target-crRNA complementarity at positions 1–5 and 7–8 of the 5′ end of the crRNA spacer sequence.
Therefore, a conserved protospacer-adjacent motif or PAM, which is present in the target, but not the CRISPR locus, allows for distinction between foreign and host DNA.
On the other hand, no PAMs have been detected for Type III CRISPR-Cas systems. The crRNA formed in the type III system contains a 5'- handle. This handle originates from the CRISPR repeat sequence, and the 3' spacer contains the target sequence. Therefore, the discrimination between self and non-self is achieved by the presence of a 5' handle, which results in “self-inactivation,” a fundamentally different process to the PAM recognition used by Type I and II systems.
Recommended by LinkedIn
PAMs also serve an additional role in the protospacer acquisition step. While studying the CRISPR-Cas system’s mechanism in Part 6, we have already discussed that Cas1 and Cas2 complex is required in capturing new protospacers from the invading viral DNA. During this acquisition process, Cas9 works with Cas1 and Cas2 to find a PAM sequence, and then Cas1 and Cas2 remove the protospacer next to it. Protospacer picking with PAMs guarantees that when the same virus infects again, and Cas9 is armed with a matching crRNA, nothing will stop it from destroying the enemy DNA.
The next part of the series is about "Why is the CRISPR-Cas system suitable for gene editing?
If you liked this article and want to know more about more about applications of CRISPR in gene editing and beyond, click the below links:
For book lovers:
For video lovers:
Happy learning!