Protospacer adjacent motif

Protospacer adjacent motif (PAM) is a DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. PAM is a component of the invading virus or plasmid, but 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.[1][2][3][4] PAM is an essential targeting component (not found in bacterial genome) which distinguishes bacterial self from non-self DNA, thereby preventing the CRISPR locus from being targeted and destroyed by nuclease.[5]

CRISPR loci in a bacterium 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"). On subsequent invasion, Cas9 nuclease attaches to tracrRNA:crRNA 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 will thus not be cut by the nuclease. But the protospacer in the invading virus or plasmid will contain the PAM sequence, and will thus be cleaved by the Cas9 nuclease.[3] For editing genes, guideRNAs (gRNAs) are synthesized to perform the function of the tracrRNA:crRNA complex in recognizing gene sequences having a PAM sequence at the 3'-end.[6][7]

The canonical PAM is the sequence 5'-NGG-3' where "N" is any nucleobase followed by two guanine ("G") nucleobases.[8] Guide RNAs (gRNAs) can transport Cas9 to anywhere 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 (designated SpCas9), whereas different PAMs are associated with the Cas9 proteins of the bacteria Neisseria meningitidis, Treponema denticola, and Streptococcus thermophilus.[9] 5'-NGA-3' can be a highly efficient non-canonical PAM for human cells, but efficiency varies with genome location.[10] Attempts have been made to engineer Cas9s to recognize different PAMs to improve ability of CRISPR-Cas9 to do gene editing at any desired genome location.[11] Cas9 of Francisella novicida recognizes the canonical PAM sequence 5'-NGG-3', but has been engineered to recognize the PAM 5'-YG-3' (where "Y" is a pyrimidine[12]), thus adding to the range of possible Cas9 targets.[13] A technology called GUIDE-Seq has been devised to assay off-target cleavages produced by such gene editing.[14] Notably, the PAM requirement can be exploited to specifically target single-nucleotide heterozygous mutations while exerting no aberrant effects on the wild-type alleles[15]

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References

  1. ↑ Mojica FJ, Díez-Villaseñor C, García-Martínez J, Almendros C (2009). "Short motif sequences determine the targets of the prokaryotic CRISPR defence system". Microbiology (journal) 155 (Pt 3): 733–740. doi:10.1099/mic.0.023960-0. PMID 19246744.
  2. ↑ Shah SA, Erdmann S, Mojica FJ, Garrett RA (2013). "Protospacer recognition motifs: mixed identities and functional diversity". RNA Biology 10 (5): 891–899. doi:10.4161/rna.23764. PMC 3737346. PMID 23403393.
  3. 1 2 Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012). "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity". Science 337 (6096): 816–821. doi:10.1126/science.1225829. PMID 22745249.
  4. ↑ Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA (2014). "DNA interrogation by the CRISPR RNA-guided endonuclease Cas9". Nature 507 (7490): 62–67. doi:10.1038/nature13011. PMC 4106473. PMID 24476820.
  5. ↑ Mali P, Esvelt KM, Church GM (2013). "Cas9 as a versatile tool for engineering biology". Nature Methods 10 (10): 957–963. doi:10.1038/nmeth.2649. PMC 4051438. PMID 24076990.
  6. ↑ Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013). "RNA-guided human genome engineering via Cas9". Science 339 (6121): 823–826. doi:10.1126/science.1232033. PMC 3712628. PMID 23287722.
  7. ↑ Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013). "Multiplex genome engineering using CRISPR/Cas systems". Science 339 (6121): 819–823. doi:10.1126/science.1231143. PMC 3795411. PMID 23287718.
  8. ↑ Anders C, Niewoehner O, Duerst A, Jinek M (2014). "Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease". Nature 513 (7519): 569–573. doi:10.1038/nature13579. PMC 4176945. PMID 25079318.
  9. ↑ Esvelt KM, Mali P, Braff JL, Moosburner M, Yaung SJ, Church GM (2013). "Orthogonal Cas9 proteins for RNA-guided gene regulation and editing". Nature Methods 10 (11): 1116–1123. doi:10.1038/nmeth.2681. PMC 3844869. PMID 24076762.
  10. ↑ Zhang Y, Ge X, Yang F, Zhang L, Zheng J, Tan X, Jin ZB, Qu J, Gu F (2014). "Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells". Scientific Reports 4: 5405. doi:10.1038/srep05405. PMC 4066725. PMID 24956376.
  11. ↑ Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, Gonzales AP, Li Z, Peterson RT, Yeh JR, Aryee MJ, Joung JK (2015). "Engineered CRISPR-Cas9 nucleases with altered PAM specificities". Nature 523 (7561): 481–485. doi:10.1038/nature14592. PMID 26098369.
  12. ↑ "Nucleotide Codes, Amino Acid Codes, and Genetic Codes". KEGG: Kyoto Encyclopedia of Genes and Genomes. July 15, 2014. Retrieved 2016-04-06.
  13. ↑ Hirano H, Gootenberg JS, Horii T, Abudayyeh OO, Kimura M, Hsu PD, Nakane T, Ishitani R, Hatada I, Zhang F, Nishimasu H, Nureki O (2016). "Structure and Engineering of Francisella novicida Cas9". Cell (journal) 164 (5): 950–961. doi:10.1016/j.cell.2016.01.039. PMID 26875867.
  14. ↑ Tsai SQ, Zheng Z, Nguyen NT, Liebers M, Topkar VV, Thapar V, Wyvekens N, Khayter C, Iafrate AJ, Le LP, Aryee MJ, Joung JK (2015). "GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleas". Nature Biotechnology 33 (2): 187–197. doi:10.1038/nbt.3117. PMC 4320685. PMID 25513782.
  15. ↑ Li Y, Mendiratta S, Ehrhardt K, Kashyap N, White MA, Bleris L. (2016). "Exploiting the CRISPR/Cas9 PAM Constraint for Single-Nucleotide Resolution Interventions". PLoS One 11 (1). PMID 26788852.
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