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Protospacer adjacent motif

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A protospacer n'akụkụ motif (PAM) bụ 2-6-base ụzọ DNA usoro ozugbo na-eso usoro DNA nke Cas9 nuclease lekwasịrị anya na CRISPR usoro mgbochi nje.[1]PAM bụ akụkụ nke nje virus ma ọ bụ plasmid na-awakpo, mana a naghị ahụ ya na mkpụrụ ndụ ihe nketa nke nje bacteria ma ya mere ọ bụghị akụkụ nke locus CRISPR nke nje bacteria. Cas9 agaghị ejikọta [2] ọ bụ kewaa usoro DNA a chọrọ ma ọ bụrụ na usoro PAM anaghị eso ya.[3][4][5] PAM [6] ihe dị mkpa nke na-eme ka ọdịiche dị n'etiti onwe nje bacteria na DNA na-abụghị onwe ya, si otú a na-egbochi CRISPR locus ka nuclease jikọtara ya na-elekwasị anya ma na-ebibi ya. [1]

Ndị na-agba ọsọ / ndị na-agba chaa chaa

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N'ime mkpụrụ ndụ nje bacteria, CRISPR loci nwere "spacers" (DNA viral nke etinyere n'ime ebe CRISPR) na n'ụdị II usoro ihe mgbochi na-eme mgbanwe ka e kere site na viral ma ọ bụ plasmid DNA (nke a na-akpọ "protospacers"). Mgbe mbuso agha na-esote, ihe jikọrọ CRISPR dị ka Cas9 na-ejikọta na tracrRNA-crRNA mgbagwoju, nke na-eduzi Cas9 na usoro protospacer na-awakpo. Mana Cas9 agaghị ekewa usoro protospacer ọ gwụla ma enwere usoro PAM dị n'akụkụ. Onye na-ahụ maka nje bacteria CRISPR loci agaghị enwe usoro PAM, ya mere, a gaghị ebipụ ya site na nuclease, ma protospacer na nje virus ma ọ bụ plasmid ga-enwe usoro PAM, ya mere, Cas9 nuclease ga-agbaji. 4] N'ime ngwa edezi genome, a na-ahazi oligonucleotide dị mkpirikpi nke a maara dị ka onye ndu RNA (gRNA) iji rụọ ọrụ nke mgbagwoju tracrRNA-crRNA n'ịghọta usoro mkpụrụ ndụ ihe nketa nwere usoro PAM na 3'-ngwụsị, si otú ahụ "na-eduzi" nuclease ka usoro a kapịrị ọnụ nke nuclease nwere ike ịkpụ.[5] [7][8]

Usoro PAM

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PAM na nha nke CRISPR DNA nucleases dị iche iche

Canonical PAM [9] usoro 5'-NGG-3', ebe "N" bụ nucleobase ọ bụla guanine ("G") nucleobases abụọ sochiri. RNA ndị nduzi nwere ike ibuga Cas9 na locus ọ bụla na genome maka nhazi mkpụrụ ndụ ihe nketa, mana enweghị nhazi nwere ike ime na saịtị ọ bụla ọzọ karịa otu Cas9 na-amata PAM. [10] na-ejikọta PAM canonical na Cas9 nuclease nke Streptococcus pyogenes (nke a họpụtara SpCas9), ebe PAM dị iche iche na-ejikarị protein Cas9 nke nje bacteria Neisseria meningitidis, Treponema denticola, na Streptococcus thermophilus. [11]'-NGA-3' nwere ike ịbụ PAM na-adịghị arụ ọrụ nke ọma maka mkpụrụ ndụ mmadụ, mana arụmọrụ dịgasị iche na ọnọdụ genome. [12] meela mgbalị iji mepụta Cas9s iji mata PAM dị iche iche iji melite ikike nke CRISPR-Cas9 iji dezie mkpụrụ ndụ ihe nketa na ebe ọ bụla achọrọ.

Cas9 nke Francisella novicida na-amata usoro PAM 5'-NGG-3', mana emeela ya iji mata 5'-YG-3' (ebe "Y" bụ pyrimidine [13]), si otú a na-agbakwunye na ọnụọgụ nke Cas9 nwere ike. [14] Cpf1 nuclease nke Francisella novicida na-amata PAM 5'-TTTN-3'[15] ma ọ bụ 5'-YTN-3'.[16]

Ewezuga CRISPR-Cas9 [17] CRISPR -Cpf1, enwere ọtụtụ nucleases na PAMs a na-achọpụtabeghị.

CRISPR/Cas13a (nke bụbu C2c2 [18]) sitere na nje bacteria Leptotrichia shahii bụ usoro CRISPR nke RNA na-eduzi nke na-elekwasị anya na usoro na RNA karịa DNA. PAM adịghị mkpa maka CRISPR [19]-elekwasị anya na RNA, ọ bụ ezie na guanine na-emetụta ịdị irè nke ọma, a họpụtakwara ya dị ka "protospacer flanking site" (PFS). [1]

GUIDE-Seq

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[20] mepụtara teknụzụ a na-akpọ GUIDE-Seq iji nyochaa nkewa ndị na-abụghị nke a na-eme site na nhazi mkpụrụ ndụ ihe nketa dị otú ahụ. Enwere ike iji ihe achọrọ PAM mee ihe iji lekwasị anya [21] otu nucleotide heterozygous mutations mgbe ọ na-enweghị mmetụta ọ bụla na ụdị ala ọhịa.

Hụkwa

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  • CRISPR
  • CRISPR/Cpf1

Njikọ mpụga

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Edensibia

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  1. ↑ (2013) "Protospacer recognition motifs: mixed identities and functional diversity". RNA Biology 10 (5): 891–899. DOI:10.4161/rna.23764. PMID 23403393. 
  2. ↑ (2014) "DNA interrogation by the CRISPR RNA-guided endonuclease Cas9". Nature 507 (7490): 62–67. DOI:10.1038/nature13011. PMID 24476820. 
  3. ↑ (2009) "Short motif sequences determine the targets of the prokaryotic CRISPR defence system". Microbiology 155 (Pt 3): 733–740. DOI:10.1099/mic.0.023960-0. PMID 19246744. 
  4. ↑ (2013) "Protospacer recognition motifs: mixed identities and functional diversity". RNA Biology 10 (5): 891–899. DOI:10.4161/rna.23764. PMID 23403393. 
  5. ↑ 5.0 5.1 (2012) "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity". Science 337 (6096): 816–821. DOI:10.1126/science.1225829. PMID 22745249. 
  6. ↑ (2013) "Cas9 as a versatile tool for engineering biology". Nature Methods 10 (10): 957–963. DOI:10.1038/nmeth.2649. PMID 24076990. 
  7. ↑ (2013) "Multiplex genome engineering using CRISPR/Cas systems". Science 339 (6121): 819–823. DOI:10.1126/science.1231143. PMID 23287718. 
  8. ↑ (2013) "RNA-guided human genome engineering via Cas9". Science 339 (6121): 823–826. DOI:10.1126/science.1232033. PMID 23287722. 
  9. ↑ (2014) "Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease". Nature 513 (7519): 569–573. DOI:10.1038/nature13579. PMID 25079318. 
  10. ↑ (2013) "Orthogonal Cas9 proteins for RNA-guided gene regulation and editing". Nature Methods 10 (11): 1116–1123. DOI:10.1038/nmeth.2681. PMID 24076762. 
  11. ↑ (2014) "Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells". Scientific Reports 4: 5405. DOI:10.1038/srep05405. PMID 24956376. 
  12. ↑ (2015) "Engineered CRISPR-Cas9 nucleases with altered PAM specificities". Nature 523 (7561): 481–485. DOI:10.1038/nature14592. PMID 26098369. 
  13. ↑ Nucleotide Codes, Amino Acid Codes, and Genetic Codes. KEGG: Kyoto Encyclopedia of Genes and Genomes (July 15, 2014). Retrieved on 2016-04-06.
  14. ↑ (2016) "Structure and Engineering of Francisella novicida Cas9". Cell 164 (5): 950–961. DOI:10.1016/j.cell.2016.01.039. PMID 26875867. 
  15. ↑ (2015) "Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system". Cell 163 (3): 759–771. DOI:10.1016/j.cell.2015.09.038. PMID 26422227. 
  16. ↑ (2016) "The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA". Nature 532 (7600): 517–521. DOI:10.1038/nature17945. PMID 27096362. 
  17. ↑ "Even CRISPR", The Economist. Retrieved on 2016-05-27.
  18. ↑ (2017) "Diversity and evolution of class 2 CRISPR-Cas systems". Nat. Rev. Microbiol. 15 (3): 169–182. DOI:10.1038/nrmicro.2016.184. PMID 28111461. 
  19. ↑ (2016) "C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector". Science 353 (6299): aaf5573. DOI:10.1126/science.aaf5573. PMID 27256883. 
  20. ↑ (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. PMID 25513782. 
  21. ↑ (2016) "Exploiting the CRISPR/Cas9 PAM Constraint for Single-Nucleotide Resolution Interventions". PLoS One 11 (1): e0144970. DOI:10.1371/journal.pone.0144970. PMID 26788852. 
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