The PAM, also known as the protospacer adjacent motif, is a short specific DNA sequence (usually 2-6 base pairs in length), that follows the DNA region targeted for cleavage by the CRISPR system, such as CRISPR-Cas9. The PAM is required for a Cas nuclease to cut and is generally found 3-4 nucleotides downstream from the cut site. The most commonly used Cas9 nuclease, derived from S. pyogenes, recognizes a PAM sequence of 5'-NGG-3' (where "N" can be any nucleotide base) that is found directly downstream of the target sequence in the genomic DNA, on the non-target strand. That is, PAM are recognized directly by the endonuclease protein, rather than its associated guide RNA. The genomic locations that can be targeted for editing by CRISPR are limited by the presence and locations of the nuclease-specific PAM sequences.
CRISPR-based immunity acts by integrating short virus sequences in the cell's CRISPR locus, allowing the cell to remember, recognize and clear infections. How does the viral genome fragment embedded in the bacterial genome not trigger the Cas nuclease to cut of the bacterial genome? The very basis of bacteria evading their own endonucleases is the PAM sequence. When the Cas nuclease cuts fragments of the viral genome and stores bits in the CRISPR repeats it excludes the PAM sequence even though it is involved in recognition to ensure that the bacterial genome is not recognized as a target. That is, PAM is a component of the invading virus or plasmid, but is not a component of the bacterial CRISPR locus. Cas nuclease will not successfully bind to or cleave the target DNA sequence if it is not followed by the PAM sequence.
Fig 1. The canonical sequence and location of the PAM.
Fig 2. PAM orientation and targets for different types of CRISPR-Cas systems.
Despite the common role of the PAM in target recognition, its characteristics vary between different types of CRISPR-Cas systems. One major difference is the location of the PAM. Using the non-target strand of the protospacer as a reference, the PAM is located on the 5′ of the protospacer for Type I and V systems and on the 3′ end of the protospacer for Type II. In the case of Type III and Type VI systems, limited evidence suggests that the PAM is located within the target RNA. Aside from location, the composition of PAM can vary widely. The composition includes the sequences comprising the PAM, the length of the linker separating the protospacer and the sequence-specific portion of the PAM, and the promiscuity in deviating from a defined consensus sequence. PAMs can vary widely even within a given subtype, more work is needed to fully interrogate the diversity of PAMs in nature.
|Cas Nucleases||Organisms||PAM Sequences (From 5' to 3')||Types|
|SpCas9||Streptococcus pyogenes||NGG or NRG||Type II-A|
|LiCas9||Listeria innocua||NGG||Type II-A|
|SaCas9||Staphylococcus aureus||NGRRT or NGRRN|
|NmCas9||Neisseria meningitidis||NNNNGATT||Type II-C|
|CjCas9||Campylobacter jejuni||NNNNRYAC||Type II-C|
|LbCpf1||Lachnospiraceae bacterium||TTTN||Type V|
|AsCpf1||Acidaminococcus sp.||TTTN||Type V|
|FnCpf1||Francisella novicida||TTN||Type V|
The most commonly-used Cas9 from Streptococcus pyogenes recognizes the PAM sequence 5′-NGG-3′, which is the canonical sequence for the PAM. There are many different Cas9 endonucleases to choose from isolated from different bacterial species, and each recognizes a different PAM. Related studies have suggested that the type II CRISPR system can also use NRG (where R is G or A) as PAM sequence, albeit with only one-ﬁfth of the binding efﬁciency compared to NGG. Several studies reported that the NRG sequence is the predominant noncanonical PAM for CRISPR-Cas9-mediated DNA cleavage at the human EMX locus. But the NRG is not the optimal PAM for the designing of CRISPR-Cas9 sequences.
Once Cas9 protein binds its guide RNA, the complex is ready to search for complementary target DNA sites. Target search and recognition require both complementary base pairing between the 20-nt spacer sequence and a protospacer in the target DNA, as well as the presence of conserved PAM sequence adjacent to the target site. Cas effector proteins directly bind the PAM sequence through protein-DNA interactions and subsequently unzip the downstream DNA sequence. The effector proteins then interrogate the extent of base pairing between one strand of the DNA target and the spacer portion of the CRISPR RNA. Sufficient complementarity between the two drives target cleavage. The PAM sequence is crucial for the discrimination between self and nonself sequences, only if the invading DNA is ﬂanked by the correct PAM can it be cleaved during interference.
Aside from its function in immune recognition, it was shown that PAMs are of critical importance for recognition and selection of protospacer during acquisition. It was found that protospacers ﬂanked by the correct PAM could be incorporated into the CRISPR array. In this case, the acquisition proteins alone or in coordination with effector proteins recognize defined PAM sequences while acquiring new spacers, ensuring that each new spacer can recognize the invading DNA. As PAMs for acquisition and interference can be different, the associated sequences have been respectively termed spacer acquisition motifs (SAMs) and target interference motifs (TIMs). While this distinction is important, we refer to both motifs as PAMs given our primary focus on immune defense and the limited adoption of the terms SAMs and TIMs.
Fig 3. Functions of the CRISPR PAM. A. CRISPR spacer acquisition; B. CRISPR interference
The ability to engineer PAM recognition holds great potential for CRISPR technologies. One potential outcome is revising the process of guide-RNA design. The current process involves identifying a PAM within a genetic locus and then selecting the flanking sequence as the target, such as the homing RNAs. The homing RNAs editing results in far more diversity in accumulated mutations relative to conventional CRISPR guides. PAM engineering could be also used to generate a collection of effector proteins that together recognize all possible PAM sequences. These mutations could be combined with those known to better reject mismatches between the guide RNA and the target, potentially yielding highly specific Cas effector proteins with negligible off-target effects. The most successful PAM engineering efforts to-date combined random mutagenesis of the DNA binding residues or the entire protein with a high-throughput dual selection.
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