CRISPR gene editing

CRISPR-Cas9

CRISPR gene editing (CRISPR, pronounced /ˈkrɪspər/ "crisper", refers to "clustered regularly interspaced short palindromic repeats") is a genetic engineering technique in molecular biology by which the genomes of living organisms may be modified. It is based on a simplified version of the bacterial CRISPR-Cas9 antiviral defense system. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added in vivo.[1]

The technique is considered highly significant in biotechnology and medicine as it enables editing genomes in vivo very precisely, cheaply, and easily. It can be used in the creation of new medicines, agricultural products, and genetically modified organisms, or as a means of controlling pathogens and pests. It also has possibilities in the treatment of inherited genetic diseases as well as diseases arising from somatic mutations such as cancer. However, its use in human germline genetic modification is highly controversial. The development of the technique earned Jennifer Doudna and Emmanuelle Charpentier the Nobel Prize in Chemistry in 2020.[2][3] The third researcher group that shared the Kavli Prize for the same discovery,[4] led by Virginijus Šikšnys, was not awarded the Nobel prize.[5][6][7]

Working like genetic scissors, the Cas9 nuclease opens both strands of the targeted sequence of DNA to introduce the modification by one of two methods. Knock-in mutations, facilitated via homology directed repair (HDR), is the traditional pathway of targeted genomic editing approaches.[1] This allows for the introduction of targeted DNA damage and repair. HDR employs the use of similar DNA sequences to drive the repair of the break via the incorporation of exogenous DNA to function as the repair template.[1] This method relies on the periodic and isolated occurrence of DNA damage at the target site in order for the repair to commence. Knock-out mutations caused by CRISPR-Cas9 result from the repair of the double-stranded break by means of non-homologous end joining (NHEJ) or POLQ/polymerase theta-mediated end-joining (TMEJ). These end-joining pathways can often result in random deletions or insertions at the repair site, which may disrupt or alter gene functionality. Therefore, genomic engineering by CRISPR-Cas9 gives researchers the ability to generate targeted random gene disruption.

While genome editing in eukaryotic cells has been possible using various methods since the 1980s, the methods employed had proven to be inefficient and impractical to implement on a large scale. With the discovery of CRISPR and specifically the Cas9 nuclease molecule, efficient and highly selective editing became possible. Cas9 derived from the bacterial species Streptococcus pyogenes has facilitated targeted genomic modification in eukaryotic cells by allowing for a reliable method of creating a targeted break at a specific location as designated by the crRNA and tracrRNA guide strands.[8] Researcher can insert Cas9 and template RNA with ease in order to silence or cause point mutations at specific loci. This has proven invaluable for quick and efficient mapping of genomic models and biological processes associated with various genes in a variety of eukaryotes. Newly engineered variants of the Cas9 nuclease that significantly reduce off-target activity have been developed.[9]

CRISPR-Cas9 genome editing techniques have many potential applications. The use of the CRISPR-Cas9-gRNA complex for genome editing[10] was the AAAS's choice for Breakthrough of the Year in 2015.[11] Many bioethical concerns have been raised about the prospect of using CRISPR for germline editing, especially in human embryos.[12] In 2023, the first drug making use of CRISPR gene editing, Casgevy, was approved for use in the United Kingdom, to cure sickle-cell disease and beta thalassemia.[13][14] Casgevy was approved for use in the United States on December 8, 2023, by the Food and Drug Administration.[15]

  1. ^ a b c 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.
  2. ^ "The Nobel Prize in Chemistry 2020". The Nobel Prize. Retrieved 2020-12-10.
  3. ^ Cohen J (October 7, 2020). "CRISPR, the revolutionary genetic "scissors," honored by Chemistry Nobel". Science. doi:10.1126/science.abf0540. S2CID 225116732.
  4. ^ Cohen J (2018-06-04). "With prestigious prize, an overshadowed CRISPR researcher wins the spotlight". Science | AAAS. Retrieved 2020-05-02.
  5. ^ Cite error: The named reference Owens 2020 was invoked but never defined (see the help page).
  6. ^ "Lithuanian scientists not awarded Nobel prize despite discovering same technology". LRT.LT. 8 October 2020.
  7. ^ Šikšnys V (2018-06-16). "Imam genų žirkles, iškerpam klaidą, ligos nelieka". Laisvės TV / Freedom TV. 12:22 minutes in. LaisvėsTV. <...>Tai mes tą savo straipsnį išsiuntėm į redakciją pirmieji, bet laimės ten daug nebuvo. Viena redakcija pasakė, kad mes net recenzentam nesiųsim. Nusiuntėm į kitą redakciją – tai jis (straipsnis) pragulėjo kažkur ant redaktoriaus stalo labai ilgai. Na ir taip galų gale išsiuntėm į trečią žurnalą ir trečias žurnalas po kelių mėnesių jį išspausdino. Bet, aišku, Berklio universiteto mokslininkams sekėsi geriau – jie išsiuntė straipsnį į žurnalą Science – jį priėmė ir išspausdino per 2 savaites. Nors iš tikro jie tą straispnį išsiuntė pora mėnesių vėliau nei mes. Retrieved 2018-06-30. <...> Well, we were who had sent the article first, but had not much of luck.
  8. ^ Zhang JH, Pandey M, Kahler JF, Loshakov A, Harris B, Dagur PK, et al. (November 2014). "Improving the specificity and efficacy of CRISPR/CAS9 and gRNA through target specific DNA reporter". Journal of Biotechnology. 189: 1–8. doi:10.1016/j.jbiotec.2014.08.033. PMC 4252756. PMID 25193712.
  9. ^ Vakulskas CA, Dever DP, Rettig GR, Turk R, Jacobi AM, Collingwood MA, et al. (August 2018). "A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells". Nature Medicine. 24 (8): 1216–1224. doi:10.1038/s41591-018-0137-0. PMC 6107069. PMID 30082871.
  10. ^ Ledford H (March 2016). "CRISPR: gene editing is just the beginning". Nature. 531 (7593): 156–159. Bibcode:2016Natur.531..156L. doi:10.1038/531156a. PMID 26961639.
  11. ^ Travis J (17 December 2015). "Breakthrough of the Year: CRISPR makes the cut". Science Magazine. American Association for the Advancement of Science.
  12. ^ Ledford H (June 2015). "CRISPR, the disruptor". Nature. 522 (7554): 20–24. Bibcode:2015Natur.522...20L. doi:10.1038/522020a. PMID 26040877.
  13. ^ "Casgevy: UK approves gene-editing drug for sickle cell". BBC News. 16 November 2023. Retrieved 16 November 2023.
  14. ^ "MHRA authorises world-first gene therapy that aims to cure sickle-cell disease and transfusion-dependent β-thalassemia". Gov.uk. 16 November 2023. Retrieved 16 November 2023.
  15. ^ "FDA Approves First Gene Therapies to Treat Patients with Sickle Cell Disease". Food and Drug Administration. 11 December 2023. Retrieved 11 December 2023.