Cas13

from Wikipedia, the free encyclopedia

Cas13 (from C RISPR- as sociated) are a class of endonucleases and thus proteins that occur as ribonucleoproteins in the adaptive immune defense of prokaryotes . They are used in molecular biology for the specific modification and detection of RNA and as an antiviral therapeutic.

properties

Cas13 belongs to the Cas proteins of class 2 type VI, which play a central role in the adaptive immune defense of prokaryotes against phages . So far, four subtypes are known. Cas13a (formerly C2c2), Cas13b, Cas13c, and Cas13d. In the case of the Cas13b subtype, a distinction is also made between the Cas13b1 and Cas13b1 variants. By incorporating specific RNA molecules called CRISPR-RNA (crRNA), the Cas13 effector complex is created, which binds and cuts complementary sections of the phage RNA. Cas13 is therefore an RNA-mediated RNA-binding nuclease . The specificity arises from a 28-30 nucleotide long sequence within the crRNA, which is referred to as a spacer. In contrast to Type II Cas proteins (such as Cas9 ), which only cut double-stranded DNA , they do not require any trans-activating crRNA (tracrRNA). The specific binding of the effector complex to the target sequence leads in some Cas13 representatives (for example Cas13a) in bacteria and in vitro to the activation of an unspecific ribonuclease activity. While DNA-binding Cas proteins always rely on a Protospacer Adjacent Motif (PAM) for binding , some Cas13 proteins require a Protospacer Flanking Site (PFS) instead of a PAM. In Cas13a from Leptotrichia shahii, this PFS consists of any nucleotide except guanosine . However, it has been shown that Cas13 proteins of other species do not require PFS for binding.

application

The ability of Cas3 proteins to specifically bind and cut RNA sequences is used in a variety of molecular biology for the detection of viruses and the modification of eukaryotic mRNA . They are also used experimentally as an antiviral therapeutic.

Virus detection

Cas13 proteins can be used to detect viruses at the nucleic acid level in the attomolar range. The system developed for this is called SHERLOCK, an acronym for “ Specific High-sensitivity Enzymatic Reporter unLOCKing ”. The system was recently adapted to detect the SARS-CoV-2 in patient samples. The system was called STOPCovid (SHERLOCK Testing in One Pot) and works with Cas12b.

Therapeutic use

The CRISPR-Cas13 system also shows potential as an antiviral therapeutic in vitro . In a first study, the virus RNA and the correspondingly transcribed mRNA were successfully degraded by Cas13 in human cells infected with SARS-CoV-2 . Cas13d from Ruminococcus flavefaciens was used for this , as this variant has a particularly high activity in human cells. The technology is called PAC-MAN (Prophylactic Antiviral CRISPR in huMAN cells) by the inventors. With the same strategy, human cells infected with the influenza A virus type H1N1 could also be treated successfully.

RNA modification in eukaryotes

Cas13a from Leptotrichia wadei (LwaCas13a; formerly C2c2) was used in mammalian and plant cells to inhibit gene expression by knockdown . In the same study, a catalytically inactive “dead” version of Cas13a (dCas13a) was also used to detect mRNA in living cells. By fusion of dCas13a with adenosine deaminase domain of ADAR2 also the mRNA was targeted modification of mammalian cells in another study nucleotides. In further studies it was shown that certain orthologues of Cas13b and Cas13d have a higher activity in mammalian cells (PspCas13b from Prevotella sp. P5-125 and RfxCas13d from Ruminococcus flavefaciens ). The relatively small protein size of Cas13d compared to other Cas13 representatives also allows its use in adeno-associated viral vectors .

Web links

  • stopcovid.science - Information about STOPCovid from the inventors of the technology

Individual evidence

  1. ^ A b Adrian Pickar-Oliver, Charles A. Gersbach: The next generation of CRISPR - Cas technologies and applications . In: Nature Reviews Molecular Cell Biology . tape 20 , no. 8 , August 2019, ISSN  1471-0072 , p. 490-507 , doi : 10.1038 / s41580-019-0131-5 , PMID 31147612 , PMC 7079207 (free full text).
  2. ↑ a b c d Timothy R. Abbott, Girija Dhamdhere, Yanxia Liu, Xueqiu Lin, Laine Goudy: Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza . In: Cell . tape 181 , no. 4 , May 2020, p. 865–876.e12 , doi : 10.1016 / j.cell.2020.04.020 , PMID 32353252 , PMC 7189862 (free full text).
  3. ↑ Sergey Shmakov, Aaron Smargon, David Scott, David Cox, Neena Pyzocha: Diversity and evolution of class 2 CRISPR-Cas systems . In: Nature Reviews Microbiology . tape 15 , no. 3 , March 2017, ISSN  1740-1526 , p. 169–182 , doi : 10.1038 / nrmicro.2016.184 , PMID 28111461 , PMC 5851899 (free full text).
  4. ↑ Kira S. Makarova, Yuri I. Wolf, Jaime Iranzo, Sergey A. Shmakov, Omer S. Alkhnbashi: Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants . In: Nature Reviews Microbiology . tape 18 , no. 2 , February 2020, ISSN  1740-1526 , p. 67-83 , doi : 10.1038 / s41579-019-0299-x .
  5. ^ Lay, Martin, Springer-Verlag GmbH: Genetics and Molecular Biology . Berlin 2017, ISBN 978-3-662-50273-0 .
  6. ↑ a b 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 ).
  7. ↑ Alexandra East-Seletsky, Mitchell R. O'Connell, Spencer C. Knight, David Burstein, Jamie HD Cate: Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection . In: Nature . tape 538 , no. 7624 , October 2016, ISSN  0028-0836 , p. 270–273 , doi : 10.1038 / nature19802 , PMID 27669025 , PMC 5576363 (free full text).
  8. ^ Mazhar Adli: The CRISPR tool kit for genome editing and beyond . In: Nature Communications . tape 9 , no. 1 , December 2018, ISSN  2041-1723 , p. 1911 , doi : 10.1038 / s41467-018-04252-2 , PMID 29765029 , PMC 5953931 (free full text).
  9. ↑ Omar O. Abudayyeh, Jonathan S. Gootenberg, Silvana Konermann, Julia Joung, Ian M. Slaymaker: C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector . In: Science . tape 353 , no. 6299 , August 5, 2016, ISSN  0036-8075 , p. aaf5573 , doi : 10.1126 / science.aaf5573 , PMID 27256883 , PMC 5127784 (free full text).
  10. ↑ 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).
  11. ↑ a b 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).
  12. ↑ Jonathan S. Gootenberg, Omar O. Abudayyeh, Jeong Wook Lee, Patrick Essletzbichler, Aaron J. Dy: Nucleic acid detection with CRISPR-Cas13a / C2c2 . In: Science . tape 356 , no. 6336 , April 28, 2017, ISSN  0036-8075 , p. 438-442 , doi : 10.1126 / science.aam9321 , PMID 28408723 , PMC 5526198 (free full text).
  13. ↑ Jonathan S. Gootenberg, Omar O. Abudayyeh, Max J. Kellner, Julia Joung, James J. Collins: Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6 . In: Science . tape 360 , no. 6387 , April 27, 2018, ISSN  0036-8075 , p. 439-444 , doi : 10.1126 / science.aaq0179 , PMID 29449508 , PMC 5961727 (free full text).
  14. ↑ Julia Joung, Alim Ladha, Makoto Saito, Michael Segel, Robert Bruneau: Point-of-care testing for COVID-19 using SHERLOCK diagnostics . May 8, 2020, doi : 10.1101 / 2020.05.04.20091231 .
  15. ↑ STOPCovid. Retrieved May 19, 2020 (English).
  16. ↑ Winston X. Yan, Shaorong Chong, Huaibin Zhang, Kira S. Makarova, Eugene V. Koonin: Cas13d Is a Compact RNA-Targeting Type VI CRISPR Effector Positively Modulated by a WYL-Domain-Containing Accessory Protein . In: Molecular Cell . tape 70 , no. 2 , April 2018, p. 327–339.e5 , doi : 10.1016 / j.molcel.2018.02.028 , PMID 29551514 , PMC 5935466 (free full text).
  17. ↑ 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).
  18. ↑ 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).
  19. ↑ Silvana Konermann, Peter Lotfy, Nicholas J. Brideau, Jennifer Oki, Maxim N. Shokhirev: Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors . In: Cell . tape 173 , no. 3 , April 2018, p. 665–676.e14 , doi : 10.1016 / j.cell.2018.02.033 , PMID 29551272 , PMC 5910255 (free full text).