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 DNA ...

DNA Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...

sequence immediately following the DNA sequence targeted by the

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

nuclease In biochemistry, a nuclease (also archaically known as nucleodepolymerase or polynucleotidase) is an enzyme capable of cleaving the phosphodiester bonds that link nucleotides together to form nucleic acids. Nucleases variously affect single and ...

in the

CRISPR CRISPR (; acronym of clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea. Each sequence within an individual prokaryotic CRISPR is d ...

bacterial

adaptive immune system The adaptive immune system (AIS), also known as the acquired immune system, or specific immune system is a subsystem of the immune system that is composed of specialized cells, organs, and processes that eliminate pathogens specifically. The ac ...

. 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– 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 insert ge ...

applications, a short oligonucleotide known as a guide RNA (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 Cas9 PAM is the sequence 5'-NGG-3', where "N" is any

nucleobase Nucleotide bases (also nucleobases, nitrogenous bases) are nitrogen-containing biological compounds that form nucleosides, which, in turn, are components of nucleotides, with all of these monomers constituting the basic building blocks of nuc ...

followed by two

guanine Guanine () (symbol G or Gua) is one of the four main nucleotide bases 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 ...

("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 ...

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

bacteria Bacteria (; : bacterium) are ubiquitous, mostly free-living organisms often consisting of one Cell (biology), biological cell. They constitute a large domain (biology), domain of Prokaryote, prokaryotic microorganisms. Typically a few micr ...

Neisseria meningitidis ''Neisseria meningitidis'', often referred to as the 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 a ...

'', '' Treponema denticola'', and ''

Streptococcus thermophilus ''Streptococcus thermophilus'' formerly known as ''Streptococcus salivarius ''subsp.'' thermophilus'' is a gram-positive bacteria, gram-positive bacterium, and a lactic acid fermentation, fermentative facultative anaerobic organism, facultative ...

''. 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'' 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 oth ...

), 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