A protospacer adjacent motif (PAM) is a 2–6-

base pair A base pair (bp) is a fundamental unit of double-stranded nucleic acids consisting of two nucleobases bound to each other by hydrogen bonds. They form the building blocks of the DNA double helix and contribute to the folded structure of both D ...

DNA sequence immediately following the DNA sequence targeted by the

Cas9 Cas9 (CRISPR associated protein 9, formerly called Cas5, Csn1, or Csx12) is a 160 kilodalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids, and is heavily utilized in gene ...

nuclease A nuclease (also archaically known as nucleodepolymerase or polynucleotidase) is an enzyme capable of cleaving the phosphodiester bonds between nucleotides of nucleic acids. Nucleases variously effect single and double stranded breaks in their t ...

in the

CRISPR CRISPR () (an acronym for clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments of bacte ...

bacterial

adaptive immune system The adaptive immune system, also known as the acquired immune system, is a subsystem of the immune system that is composed of specialized, systemic cells and processes that eliminate pathogens or prevent their growth. The acquired immune system ...

. The PAM is a component of the invading virus or plasmid, but is not found in the bacterial host genome and hence is not a component of the bacterial CRISPR locus. Cas9 will not successfully bind to or cleave the target DNA sequence if it is not followed by the PAM sequence. PAM is an essential targeting component which distinguishes bacterial self from non-self DNA, thereby preventing the CRISPR locus from being targeted and destroyed by the CRISPR-associated nuclease.

Spacers/protospacers

In a bacterial genome, CRISPR loci contain "spacers" (viral DNA inserted into a CRISPR locus) that in type II adaptive immune systems were created from invading viral or plasmid DNA (called "protospacers"). Upon subsequent invasion, a CRISPR-associated

such as

attaches to a

tracrRNA In molecular biology, trans-activating crispr RNA (tracrRNA) is a small ''trans''-encoded RNA. It was first discovered by Emmanuelle Charpentier in her study of human pathogen ''Streptococcus pyogenes'', a type of bacteria that causes harm to human ...

– crRNA complex, which guides Cas9 to the invading protospacer sequence. But Cas9 will not cleave the protospacer sequence unless there is an adjacent PAM sequence. The spacer in the bacterial CRISPR loci will not contain a PAM sequence, and thus will not be cut by the nuclease, but the protospacer in the invading virus or plasmid will contain the PAM sequence, and thus will be cleaved by the Cas9 nuclease. In

genome editing Genome editing, or genome engineering, or gene editing, is a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Unlike early genetic engineering techniques that randomly inserts ...

applications, a short oligonucleotide known as a

guide RNA A guide RNA (gRNA) is a piece of RNA that functions as a guide for RNA- or DNA-targeting enzymes, with which it forms complexes. Very often these enzymes will delete, insert or otherwise alter the targeted RNA or DNA. They occur naturally, serv ...

(gRNA) is synthesized to perform the function of the tracrRNA–crRNA complex in recognizing gene sequences having a PAM sequence at the

3'-end Directionality, in molecular biology and biochemistry, is the end-to-end chemical orientation of a single strand of nucleic acid. In a single strand of DNA or RNA, the chemical convention of naming carbon atoms in the nucleotide pentose-sugar-ri ...

, thereby "guiding" the nuclease to a specific sequence which the nuclease is capable of cutting.

PAM sequences

The canonical PAM is the sequence 5'-NGG-3', where "N" is any

nucleobase Nucleobases, also known as ''nitrogenous bases'' or often simply ''bases'', are nitrogen-containing biological compounds that form nucleosides, which, in turn, are components of nucleotides, with all of these monomers constituting the basic b ...

followed by two

guanine Guanine () ( symbol G or Gua) is one of the four main nucleobases found in the nucleic acids DNA and RNA, the others being adenine, cytosine, and thymine (uracil in RNA). In DNA, guanine is paired with cytosine. The guanine nucleoside is called ...

("G") nucleobases. Guide RNAs can transport Cas9 to any locus in the genome for gene editing, but no editing can occur at any site other than one at which Cas9 recognizes PAM. The canonical PAM is associated with the Cas9 nuclease of ''

Streptococcus pyogenes ''Streptococcus pyogenes'' is a species of Gram-positive, aerotolerant bacteria in the genus ''Streptococcus''. These bacteria are extracellular, and made up of non-motile and non-sporing cocci (round cells) that tend to link in chains. They are ...

'' (designated SpCas9), whereas different PAMs are associated with the Cas9 proteins of the

bacteria Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were among ...

Neisseria meningitidis ''Neisseria meningitidis'', often referred to as meningococcus, is a Gram-negative bacterium that can cause meningitis and other forms of meningococcal disease such as meningococcemia, a life-threatening sepsis. The bacterium is referred to as a ...

'', ''

Treponema denticola ''Treponema denticola'' is a Gram-negative, obligate anaerobic, motile and highly proteolytic spirochete bacterium. It is one of four species of oral spirochetes to be reliably cultured, the others being ''Treponema pectinovorum, Treponema socra ...

'', and ''

Streptococcus thermophilus ''Streptococcus thermophilus'' also known as ''Streptococcus salivarius ''subsp.'' thermophilus'' is a gram-positive bacterium, and a fermentative facultative anaerobe, of the '' viridans'' group. It tests negative for cytochrome, oxidase, and ...

''. 5'-NGA-3' can be a highly efficient non-canonical PAM for human cells, but efficiency varies with genome location. Attempts have been made to engineer Cas9s to recognize different PAMs in order to improve the ability of CRISPR-Cas9 to edit genes at any desired genome location. The Cas9 of ''

Francisella novicida ''Francisella novicida'' is a bacterium of the Francisellaceae family, which consist of Gram-negative pathogenic bacteria. These bacteria vary from small cocci to rod-shaped, and are most known for their intracellular parasitic capabilities. In t ...

'' recognizes the canonical PAM sequence 5'-NGG-3', but has been engineered to recognize 5'-YG-3' (where "Y" is a

pyrimidine Pyrimidine (; ) is an aromatic, heterocyclic, organic compound similar to pyridine (). One of the three diazines (six-membered heterocyclics with two nitrogen atoms in the ring), it has nitrogen atoms at positions 1 and 3 in the ring. The other ...

), thus adding to the range of possible Cas9 targets. The Cpf1 nuclease of ''Francisella novicida'' recognizes the PAM 5'-TTTN-3' or 5'-YTN-3'. Aside from CRISPR-Cas9 and CRISPR-Cpf1, there are doubtless many yet undiscovered nucleases and PAMs. CRISPR/Cas13a (formerly C2c2) from the bacterium '' Leptotrichia shahii'' is an RNA-guided CRISPR system that targets sequences in RNA rather than DNA. PAM is not relevant for an RNA-targeting CRISPR, although a guanine flanking the target negatively affects efficacy, and has been designated a "protospacer flanking site" (PFS).

GUIDE-Seq

A technology called GUIDE-Seq has been devised to assay off-target cleavages produced by such gene editing. The PAM requirement can be exploited to specifically target single-nucleotide heterozygous mutations while exerting no aberrant effects on wild-type alleles.

External links

Addgene CRISPR-Cas Guide

References

{{reflist Genome editing

Spacers/protospacers

PAM sequences

GUIDE-Seq

See also

External links

References