CRISPR-Cas9

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A CRISPR-Cas9 is a CRISPR system that is from Streptococcus pyogenes and relies on the protein Cas9.

References

2023a

  • (Wikipedia, 2023) ⇒ https://en.wikipedia.org/wiki/CRISPR Retrieved:2023-1-15.
    • 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.[1] These sequences are derived from DNA fragments of bacteriophages that had previously infected the prokaryote. They are used to detect and destroy DNA from similar bacteriophages during subsequent infections. Hence these sequences play a key role in the antiviral (i.e. anti-phage) defense system of prokaryotes and provide a form of acquired immunity.[1] [2] [3] CRISPR is found in approximately 50% of sequenced bacterial genomes and nearly 90% of sequenced archaea.[4]

       Cas9 (or "CRISPR-associated protein 9") is an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence. Cas9 enzymes together with CRISPR sequences form the basis of a technology known as CRISPR-Cas9 that can be used to edit genes within organisms. [5] This editing process has a wide variety of applications including basic biological research, development of biotechnological products, and treatment of diseases. [6] [7] The development of the CRISPR-Cas9 genome editing technique was recognized by the Nobel Prize in Chemistry in 2020 which was awarded to Emmanuelle Charpentier and Jennifer Doudna.[8] [9]

      In 2022, in a proceeding at the United States Patent and Trademark Office (interference 106,115), the Patent Trial and Appeal Board decided that the inventor for the US patent covering application of CRISPR-Cas9 in eukaryotic cells is Feng Zhang, a professor of the Broad Institute.

  1. ↑ 1.0 1.1 Barrangou R (2015). “The roles of CRISPR-Cas systems in adaptive immunity and beyond". Current Opinion in Immunology. 32: 36–41. doi:10.1016/j.coi.2014.12.008. PMID 25574773.
  2. ↑ Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, et al. (March 2007). “CRISPR provides acquired resistance against viruses in prokaryotes". Science. 315 (5819): 1709–1712. Bibcode:2007Sci...315.1709B. doi:10.1126/science.1138140. hdl:20.500.11794/38902. PMID 17379808. S2CID 3888761.
  3. ↑ Marraffini LA, Sontheimer EJ (December 2008). "CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA". Science. 322 (5909): 1843–1845. Bibcode:2008Sci...322.1843M. doi:10.1126/science.1165771. PMC 2695655. PMID 19095942.
  4. ↑ https://ui.adsabs.harvard.edu/abs/2007Sci...315.1709B
  5. ↑ Bak RO, Gomez-Ospina N, Porteus MH (August 2018). “Gene Editing on Center Stage". Trends in Genetics. 34 (8): 600–611. doi:10.1016/j.tig.2018.05.004. PMID 29908711. S2CID 49269023.
  6. ↑ CRISPR-CAS9, TALENS and ZFNS - the battle in gene editing https://www.ptglab.com/news/blog/crispr-cas9-talens-and-zfns-the-battle-in-gene-editing/
  7. ↑ Hsu PD, Lander ES, Zhang F (June 2014). "Development and applications of CRISPR-Cas9 for genome engineering". Cell. 157 (6): 1262–1278. doi:10.1016/j.cell.2014.05.010. PMC 4343198. PMID 24906146.
  8. ↑ "Press release: The Nobel Prize in Chemistry 2020". Nobel Foundation. Retrieved 7 October 2020.
  9. ↑ Wu KJ, Peltier E (7 October 2020). "Nobel Prize in Chemistry Awarded to 2 Scientists for Work on Genome Editing – Emmanuelle Charpentier and Jennifer A. Doudna developed the Crispr tool, which can alter the DNA of animals, plants and microorganisms with high precision". The New York Times. Retrieved 7 October 2020.

2023b

  • (Wikipedia, 2023) ⇒ https://en.wikipedia.org/wiki/CRISPR#Cas9 Retrieved:2023-1-15.
    • A simpler CRISPR system from Streptococcus pyogenes relies on the protein Cas9. The Cas9 endonuclease is a four-component system that includes two small molecules: crRNA and trans-activating CRISPR RNA (tracrRNA).[1] [2] Jennifer Doudna and Emmanuelle Charpentier re-engineered the Cas9 endonuclease into a more manageable two-component system by fusing the two RNA molecules into a "single-guide RNA" that, when combined with Cas9, could find and cut the DNA target specified by the guide RNA. This contribution was so significant that it was recognized by the Nobel Prize in Chemistry in 2020. By manipulating the nucleotide sequence of the guide RNA, the artificial Cas9 system could be programmed to target any DNA sequence for cleavage.[3] Another group of collaborators comprising Virginijus Šikšnys together with Gasiūnas, Barrangou, and Horvath showed that Cas9 from the S. thermophilus CRISPR system can also be reprogrammed to target a site of their choosing by changing the sequence of its crRNA. These advances fueled efforts to edit genomes with the modified CRISPR-Cas9 system.[4]

      Groups led by Feng Zhang and George Church simultaneously published descriptions of genome editing in human cell cultures using CRISPR-Cas9 for the first time.[5] [6] [7] It has since been used in a wide range of organisms, including baker's yeast (Saccharomyces cerevisiae),[8] [9] [10] the opportunistic pathogen Candida albicans,[11] zebrafish (Danio rerio), fruit flies (Drosophila melanogaster), ants (Harpegnathos saltator and Ooceraea biroi ), mosquitoes (Aedes aegypti ), nematodes (Caenorhabditis elegans),[12] plants, mice (Mus musculus domesticus),[13] [14] monkeys and human embryos.[15]

      CRISPR has been modified to make programmable transcription factors that allows targeting and activation or silencing specific genes. The CRISPR-Cas9 system has shown to make effective gene edits in Human tripronuclear zygotes first described in a 2015 paper by Chinese scientists P. Liang and Y. Xu. The system made a successful cleavage of mutant Beta-Hemoglobin (HBB) in 28 out of 54 embryos. 4 out of the 28 embryos were successfully recombined using a donor template given by the scientists. The scientists showed that during DNA recombination of the cleaved strand, the homologous endogenous sequence HBD competes with the exogenous donor template. DNA repair in human embryos is much more complicated and particular than in derived stem cells.

  1. ↑ Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, Eckert MR, Vogel J, Charpentier E (March 2011). "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III". Nature. 471 (7340): 602–607. Bibcode:2011Natur.471..602D. doi:10.1038/nature09886. PMC 3070239. PMID 21455174.
  2. ↑ Barrangou R (November 2015). "Diversity of CRISPR-Cas immune systems and molecular machines". Genome Biology. 16: 247. doi:10.1186/s13059-015-0816-9. PMC 4638107. PMID 26549499.
  3. ↑ Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (August 2012). "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity". Science. 337 (6096): 816–821. Bibcode:2012Sci...337..816J. doi:10.1126/science.1225829. PMC 6286148. PMID 22745249.
  4. ↑ Mojica FJ, Montoliu L (2016). “On the Origin of CRISPR-Cas Technology: From Prokaryotes to Mammals". Trends in Microbiology. 24 (10): 811–820. doi:10.1016/j.tim.2016.06.005. PMID 27401123.
  5. ↑ Hsu PD, Lander ES, Zhang F (June 2014). "Development and applications of CRISPR-Cas9 for genome engineering". Cell. 157 (6): 1262–1278. doi:10.1016/j.cell.2014.05.010. PMC 4343198. PMID 24906146.
  6. ↑ Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (February 2013). "Multiplex genome engineering using CRISPR/Cas systems". Science. 339 (6121): 819–823. Bibcode:2013Sci...339..819C. doi:10.1126/science.1231143. PMC 3795411. PMID 23287718.
  7. ↑ Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (February 2013). "RNA-guided human genome engineering via Cas9". Science. 339 (6121): 823–826. Bibcode:2013Sci...339..823M. doi:10.1126/science.1232033. PMC 3712628. PMID 23287722
  8. ↑ DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM (April 2013). "Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems". Nucleic Acids Research. 41 (7): 4336–4343. doi:10.1093/nar/gkt135. PMC 3627607. PMID 23460208.
  9. ↑ Zhang GC, Kong II, Kim H, Liu JJ, Cate JH, Jin YS (December 2014). "Construction of a quadruple auxotrophic mutant of an industrial polyploid saccharomyces cerevisiae strain by using RNA-guided Cas9 nuclease". Applied and Environmental Microbiology. 80 (24): 7694–7701.
  10. ↑ Liu JJ, Kong II, Zhang GC, Jayakody LN, Kim H, Xia PF, Kwak S, Sung BH, Sohn JH, Walukiewicz HE, Rao CV, Jin YS (April 2016). "Metabolic Engineering of Probiotic Saccharomyces boulardii". Applied and Environmental Microbiology. 82 (8): 2280–2287. Bibcode:2016ApEnM..82.2280L. doi:10.1128/AEM.00057-16. PMC 4959471. PMID 26850302.
  11. ↑ Vyas VK, Barrasa MI, Fink GR (2015). "Candida albicans CRISPR system permits genetic engineering of essential genes and gene families". Science Advances. 1 (3): e1500248. Bibcode:2015SciA....1E0248V. doi:10.1126/sciadv.1500248. PMC 4428347. PMID 25977940.
  12. ↑ Friedland AE, Tzur YB, Esvelt KM, Colaiácovo MP, Church GM, Calarco JA (August 2013). "Heritable genome editing in C. elegans via a CRISPR-Cas9 system". Nature Methods. 10 (8): 741–743. doi:10.1038/nmeth.2532. PMC 3822328. PMID 23817069.
  13. ↑ Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R (May 2013)."One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering". Cell. 153 (4): 910–918. doi:10.1016/j.cell.2013.04.025. PMC 3969854. PMID 23643243.
  14. ↑ Soni D, Wang DM, Regmi SC, Mittal M, Vogel SM, Schlüter D, Tiruppathi C (May 2018). "Deubiquitinase function of A20 maintains and repairs endothelial barrier after lung vascular injury". Cell Death Discovery. 4 (60): 60. doi:10.1038/s41420-018-0056-3. PMC 5955943. PMID 29796309.
  15. ↑ Baltimore D, Berg P, Botchan M, Carroll D, Charo RA, Church G, Corn JE, Daley GQ, Doudna JA, Fenner M, Greely HT, Jinek M, Martin GS, Penhoet E, Puck J, Sternberg SH, Weissman JS, Yamamoto KR (April 2015). "Biotechnology. A prudent path forward for genomic engineering and germline gene modification". Science. 348 (6230): 36–38. Bibcode:2015Sci...348...36B. doi:10.1126/science.aab1028. PMC 4394183. PMID 25791083.

2023c

  • (Wikipedia, 2023) ⇒ https://en.wikipedia.org/wiki/Cas9 Retrieved:2023-1-15.
    • 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 genetic engineering applications. Its main function is to cut DNA and thereby alter a cell's genome. The CRISPR-Cas9 genome editing technique was a significant contributor to the Nobel Prize in Chemistry in 2020 being awarded to Emmanuelle Charpentier and Jennifer Doudna.

      More technically, Cas9 is a dual RNA-guided DNA endonuclease enzyme associated with the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) adaptive immune system in Streptococcus pyogenes.[1] [2] S. pyogenes utilizes CRISPR to memorize and Cas9 to later interrogate and cleave foreign DNA, such as invading bacteriophage DNA or plasmid DNA.[2] [3] [4] Cas9 performs this interrogation by unwinding foreign DNA and checking for sites complementary to the 20 nucleotide spacer region of the guide RNA (gRNA). If the DNA substrate is complementary to the guide RNA, Cas9 cleaves the invading DNA. In this sense, the CRISPR-Cas9 mechanism has a number of parallels with the RNA interference (RNAi) mechanism in eukaryotes.

      Apart from its original function in bacterial immunity, the Cas9 protein has been heavily utilized as a genome engineering tool to induce site-directed double-strand breaks in DNA. These breaks can lead to gene inactivation or the introduction of heterologous genes through non-homologous end joining and homologous recombination respectively in many laboratory model organisms. Alongside zinc finger nucleases and transcription activator-like effector nuclease (TALEN) proteins, Cas9 is becoming a prominent tool in the field of genome editing.

      Cas9 has gained traction in recent years because it can cleave nearly any sequence complementary to the guide RNA.[2] Because the target specificity of Cas9 stems from the guide RNA:DNA complementarity and not modifications to the protein itself (like TALENs and zinc fingers), engineering Cas9 to target new DNA is straightforward. Versions of Cas9 that bind but do not cleave cognate DNA can be used to locate transcriptional activator or repressors to specific DNA sequences in order to control transcriptional activation and repression. [5] Native Cas9 requires a guide RNA composed of two disparate RNAs that associate – the CRISPR RNA (crRNA), and the trans-activating crRNA (tracrRNA).[1] Cas9 targeting has been simplified through the engineering of a chimeric single guide RNA (chiRNA). Scientists have suggested that Cas9-based gene drives may be capable of editing the genomes of entire populations of organisms.[6] In 2015, Cas9 was used to modify the genome of human embryos for the first time.

  1. ↑ 1.0 1.1 Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, Eckert MR, Vogel J, Charpentier E (March 2011). "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III". Nature. 471 (7340): 602–607. Bibcode:2011Natur.471..602D. doi:10.1038/nature09886. PMC 3070239. PMID 21455174
  2. ↑ 2.0 2.1 2.2 Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (August 2012). "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity". Science. 337 (6096): 816–21. Bibcode:2012Sci...337..816J. doi:10.1126/science.1225829. PMC 6286148. PMID 22745249.
  3. ↑ Heler R, Samai P, Modell JW, Weiner C, Goldberg GW, Bikard D, Marraffini LA (March 2015). "Cas9 specifies functional viral targets during CRISPR-Cas adaptation". Nature. 519 (7542): 199–202. Bibcode:2015Natur.519..199H. doi:10.1038/nature14245. PMC 4385744. PMID 25707807.
  4. ↑ Garneau JE, Dupuis MÈ, Villion M, Romero DA, Barrangou R, Boyaval P, Fremaux C, Horvath P, Magadán AH, Moineau S (November 2010). “The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA". Nature. 468 (7320): 67–71. Bibcode:2010Natur.468...67G. CiteSeerX 10.1.1.451.9645. doi:10.1038/nature09523. PMID 21048762. S2CID 205222849.
  5. ↑ Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, Stern-Ginossar N, Brandman O, Whitehead EH, Doudna JA, Lim WA, Weissman JS, Qi LS (July 2013). "CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes". Cell. 154 (2): 442–51. doi:10.1016/j.cell.2013.06.044. PMC 3770145. PMID 23849981.
  6. ↑ Esvelt KM, Smidler AL, Catteruccia F, Church GM (July 2014). "Concerning RNA-guided gene drives for the alteration of wild populations". eLife. 3. doi:10.7554/eLife.03401. PMC 4117217. PMID 25035423.
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