Cas9

CRISPR-associated endonuclease Cas9
S. pyogenes Cas9 in complex with sgRNA and its target DNA. PDB: 4OO8​ [1]
Identifiers
OrganismStreptococcus pyogenes M1
Symbolcas9
Alt. symbolsSpCas9
Entrez901176
PDB4OO8
RefSeq (mRNA)NC_002737.2
RefSeq (Prot)NP_269215.1
UniProtQ99ZW2
Other data
EC number3.1.-.-
ChromosomeGenomic: 0.85 - 0.86 Mb
Search for
StructuresSwiss-model
DomainsInterPro
Cas9
Identifiers
Symbol?
InterProIPR028629

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.[2]

More technically, Cas9 is a RNA-guided DNA endonuclease enzyme associated with the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) adaptive immune system in Streptococcus pyogenes.[3][4][5] S. pyogenes utilizes CRISPR to memorize and Cas9 to later interrogate and cleave foreign DNA, such as invading bacteriophage DNA or plasmid DNA.[4][6][7][8] 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. Research on the development of various cas9 variants has been a promising way of overcoming the limitation of the CRISPR-Cas9 genome editing. Some examples include Cas9 nickase (Cas9n), a variant that induces single-stranded breaks (SSBs) or variants recognizing different PAM sequences.[9] 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.[4] 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.[10] 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.[11][12] Native Cas9 requires a guide RNA composed of two disparate RNAs that associate – the CRISPR RNA (crRNA), and the trans-activating crRNA (tracrRNA).[3] 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.[13] In 2015, Cas9 was used to modify the genome of human embryos for the first time.[14]

  1. ^ Cite error: The named reference P24529477 was invoked but never defined (see the help page).
  2. ^ "The Nobel Prize in Chemistry 2020". NobelPrize.org. Retrieved 2020-10-07.
  3. ^ a b 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.
  4. ^ a b c 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.
  5. ^ Oh HS, Diaz FM, Zhou C, Carpenter N, Knipe DM (2022-01-01). "CRISPR-Cas9 Expressed in Stably Transduced Cell Lines Promotes Recombination and Selects for Herpes Simplex Virus Recombinants". Current Research in Virological Science. 3: 100023. doi:10.1016/j.crviro.2022.100023. PMC 9629518. PMID 36330462.
  6. ^ 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.
  7. ^ 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): 1709–12. Bibcode:2007Sci...315.1709B. doi:10.1126/science.1138140. hdl:20.500.11794/38902. PMID 17379808. S2CID 3888761.
  8. ^ Cite error: The named reference Garneau2010 was invoked but never defined (see the help page).
  9. ^ Uddin, Fathema; M. Rudin, Charles; Sen, Triparna (August 2020). "CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future". Frontiers in Oncology. 10: 1387. doi:10.3389/fonc.2020.01387. PMC 7427626. PMID 32850447.
  10. ^ Mali P, Esvelt KM, Church GM (October 2013). "Cas9 as a versatile tool for engineering biology". Nature Methods. 10 (10): 957–63. doi:10.1038/nmeth.2649. PMC 4051438. PMID 24076990.
  11. ^ Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, Yang L, Church GM (September 2013). "CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering". Nature Biotechnology. 31 (9): 833–8. doi:10.1038/nbt.2675. PMC 3818127. PMID 23907171.
  12. ^ 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.
  13. ^ 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.
  14. ^ Cyranoski D, Reardon S (22 April 2015). "Chinese scientists genetically modify human embryos". Nature. doi:10.1038/nature.2015.17378. S2CID 87604469.