Protospacer Adjacent Motif

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Protospacer adjacent motif (PAM, 'pre-spacer-adjacent motif') is a sequence motif of DNA in biochemistry to which the endonucleases of the Cas type bind.

properties

The PAM consists of 2 to 6 nucleotides . The PAM is the binding site for Cas on the DNA to be cut. Cas9 occurs both in the antiviral defense against bacteria ( CRISPR ) and in the course of the CRISPR / Cas method for genome editing . Without a PAM behind the sequence to be cut, the DNA will not be cut. In bacterial genomes , the PAM does not occur in combination with recognition sequences, which is why it is used in bacteria to recognize foreign DNA. The sequence bound by Cas9 from Streptococcus pyogenes is 5'-NGG-3 ', with N as any nucleic base followed by two guanines . The sequence of the PAM differs between the Cas9 variants Streptococcus pyogenes and Neisseria meningitidis , Treponema denticola and Streptococcus thermophilus . In addition, a cut can be made before the sequence 5'-NGA-3 'with A as adenosine . The PAM for Cas12b from Alicyclobacillus acidoterrestris has the sequence 5'-TTN-3 '.

Protein engineering approaches to extend the substrate specificity of Cas9 by changing the PAM binding site were investigated. The Cas9 of Francisella novicida was modified so that the PAM 5'-YG-3 'with Y is recognized as any pyrimidine and a cut is made. The Cpf1 of Francisella novicida binds the 5'-PAM TTTN-3 'where T is thymidine or 5'-YTN-3'. Cas13 proteins, such as Cas13a from Leptotrichia shahii (formerly C2c2), bind and cut RNA instead of DNA and some bind to a Protospacer Flanking Site (PFS) instead of a PAM. In Cas13a from Leptotrichia shahii, for example, this PFS consists of any nucleotide other than guanosine . However, it has been shown that Cas13 proteins of other species do not require PFS for binding.

Individual evidence

  1. ^ SA Shah, S. Erdmann, FJ Mojica, RA Garrett: Protospacer recognition motifs: mixed identities and functional diversity. In: RNA biology. Volume 10, number 5, May 2013, pp. 891-899, doi : 10.4161 / rna.23764 , PMID 23403393 , PMC 3737346 (free full text).
  2. ↑ Mojica FJ, Díez-Villaseñor C, García-Martínez J, Almendros C: Short motif sequences determine the targets of the prokaryotic CRISPR defense system . In: Microbiology . 155, No. Pt 3, 2009, pp. 733-740. doi : 10.1099 / mic.0.023960-0 . PMID 19246744 .
  3. ↑ Shah SA, Erdmann S, Mojica FJ, Garrett RA: Protospacer recognition motifs: mixed identities and functional diversity Archived from the original on September 4, 2014. Information: The archive link was inserted automatically and has not yet been checked. Please check the original and archive link according to the instructions and then remove this notice. In: RNA Biology . 10, No. 5, 2013, pp. 891-899. doi : 10.4161 / rna.23764 . PMID 23403393 . PMC 3737346 (free full text). Retrieved October 23, 2017. @1@ 2Template: Webachiv / IABot / www.landesbioscience.com
  4. ↑ Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E: A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity . In: Science . 337, No. 6096, 2012, pp. 816-821. doi : 10.1126 / science.1225829 . PMID 22745249 .
  5. ↑ Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA: DNA interrogation by the CRISPR RNA-guided endonuclease Cas9 . In: Nature . 507, No. 7490, 2014, pp. 62-67. doi : 10.1038 / nature13011 . PMID 24476820 . PMC 4106473 (free full text).
  6. ↑ Mali P, Esvelt KM, Church GM: Cas9 as a versatile tool for engineering biology . In: Nature Methods . 10, No. 10, 2013, pp. 957-963. doi : 10.1038 / nmeth.2649 . PMID 24076990 . PMC 4051438 (free full text).
  7. ↑ Anders C, Niewoehner O, Duerst A, Jinek M: Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease . In: Nature . 513, No. 7519, 2014, pp. 569-573. doi : 10.1038 / nature13579 . PMID 25079318 . PMC 4176945 (free full text).
  8. ↑ Esvelt KM, Mali P, Braff JL, Moosburner M, Yaung SJ, Church GM: Orthogonal Cas9 proteins for RNA-guided gene regulation and editing . In: Nature Methods . 10, No. 11, 2013, pp. 1116-1123. doi : 10.1038 / nmeth.2681 . PMID 24076762 . PMC 3844869 (free full text).
  9. ↑ Zhang Y, Ge X, Yang F, Zhang L, Zheng J, Tan X, Jin ZB, Qu J, Gu F: Comparison of non-canonical PAMs for CRISPR / Cas9-mediated DNA cleavage in human cells . In: Scientific Reports . 4, 2014, p. 5405. doi : 10.1038 / srep05405 . PMID 24956376 . PMC 4066725 (free full text).
  10. ↑ cas12b - CRISPR-associated endonuclease Cas12b - Alicyclobacillus acidoterrestris (strain ATCC 49025 / DSM 3922 / CIP 106132 / NCIMB 13137 / GD3B) - cas12b gene. In: uniprot.org. October 16, 2013, accessed January 24, 2019 .
  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: Engineered CRISPR-Cas9 nucleases with altered PAM specificities . In: Nature . 523, No. 7561, 2015, pp. 481-485. doi : 10.1038 / nature14592 . PMID 26098369 . PMC 4540238 (free full text).
  12. ^ Nucleotide Codes, Amino Acid Codes, and Genetic Codes . KEGG: Kyoto Encyclopedia of Genes and Genomes. July 15, 2014. Retrieved April 6, 2016.
  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: Structure and Engineering of Francisella novicida Cas9 . In: Cell . 164, No. 5, 2016, pp. 950-961. doi : 10.1016 / j.cell.2016.01.039 . PMID 26875867 . PMC 4899972 (free full text).
  14. ↑ Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F: Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system . In: Cell . 163, No. 3, 2015, pp. 759-771. doi : 10.1016 / j.cell.2015.09.038 . PMID 26422227 .
  15. ↑ Fonfara I, Richter H, Bratovič M, Le Rhun A, Charpentier E: The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA . In: Nature . 532, No. 7600, 2016, pp. 517-521. doi : 10.1038 / nature17945 . PMID 27096362 .
  16. ↑ Abudayyeh OO, Gootenberg JS, Konermann S, Joung J, Slaymaker IM, Cox DB, Shmakov S, Makarova KS, Semenova E, Minakhin L, Severinov K, Regev A, Lander ES, Koonin EV, Zhang F: C2c2 is a single -component programmable RNA-guided RNA-targeting CRISPR effector . In: Science . 2016. doi : 10.1126 / science.aaf5573 . PMID 27256883 .
  17. ↑ Kira S. Makarova, Feng Zhang, Eugene V. Koonin: SnapShot: Class 2 CRISPR-Cas Systems . In: Cell . tape 168 , no. 1-2 , January 2017, pp. 328–328.e1 , doi : 10.1016 / j.cell.2016.12.038 ( elsevier.com [accessed May 17, 2020]).
  18. ↑ Omar O. Abudayyeh, Jonathan S. Gootenberg, Patrick Essletzbichler, Shuo Han, Julia Joung: RNA targeting with CRISPR-Cas13 . In: Nature . tape 550 , no. 7675 , October 2017, ISSN  0028-0836 , p. 280–284 , doi : 10.1038 / nature24049 , PMID 28976959 , PMC 5706658 (free full text) - ( nature.com [accessed May 17, 2020]).
  19. ↑ David BT Cox, Jonathan S. Gootenberg, Omar O. Abudayyeh, Brian Franklin, Max J. Kellner: RNA editing with CRISPR-Cas13 . In: Science . tape 358 , no. 6366 , November 24, 2017, ISSN  0036-8075 , p. 1019-1027 , doi : 10.1126 / science.aaq0180 , PMID 29070703 , PMC 5793859 (free full text) - ( sciencemag.org [accessed May 17, 2020]).