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CRISPR

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Adaptive immune systems of bacteria and archaea, and the genome-editing tools made from them

CRISPR (clustered regularly interspaced short palindromic repeats) is a family of adaptive immune systems found in bacteria and archaea. A cell that survives a virus keeps a short piece of the invader's DNA in a repeated array in its own genome, makes RNA copies of those pieces, and uses them to guide a nuclease to the same sequence on the next infection.

The nuclease called Cas9 is the best known. Its guide is a short RNA that can be written to order, and so Cas9 became the most widely used tool for cutting a chosen sequence in the DNA of living cells, and in December 2023 the first therapy built on it was approved in the United States.[1]

Discovery

The first hint came in 1987. A Japanese group sequencing the iap gene of Escherichia coli, which has to do with alkaline phosphatase, found an unusual repeated sequence next to it and could not say what it was for.[2][3] Similar repeats turned up in other bacteria and in halophilic archaea. In 2002 a comparison of genomes found the family in both domains of prokaryotes, gave it the name CRISPR, and noticed four genes that always sat beside a repeat array. It called them cas genes.[4] The meaning of the spacers between the repeats arrived in 2005, when they were traced to bacteriophages and conjugative plasmids, and the strains that carried them resisted those same elements.[5] In 2007 researchers working with the dairy bacterium Streptococcus thermophilus infected it with phage, watched it add new spacers cut from the phage genome, and showed that adding or removing a spacer changed which phages the cell resisted.[6] Three years later the same species showed that its system cleaves double-stranded DNA of phage and plasmid inside the matching stretch, at specific sites.[7]

Mechanism

In the type II system, the CRISPR array is transcribed and cut into short guide RNAs, each carrying one spacer, which pair with a second small RNA, the tracrRNA.[8] The pair binds Cas9. The complex scans DNA for a short motif next to the target, called the protospacer adjacent motif, and where it finds one it checks the guide against the neighboring sequence. When they match, two domains of the protein cut the DNA: HNH cuts the strand that pairs with the guide, and RuvC-like cuts the other. The result is a double-stranded break.

In 2012 a team of structural biologists and microbiologists showed in a test tube that the two RNAs could be fused into one chimera that still directed Cas9 to a chosen site, and wrote that the finding "highlights the potential to exploit the system for RNA-programmable genome editing".[9] A group in Vilnius, working in parallel, reported that Cas9 with its guide cuts DNA at a specific site.[10]

Emmanuelle Charpentier and Jennifer Doudna shared the Nobel Prize in Chemistry in 2020 "for the development of a method for genome editing".[11]

Editing

Two papers in Science in January 2013 moved the system into human cells. One, from the laboratory of George Church, used custom guide RNA to edit the AAVS1 locus, with targeting rates of 10 to 25% in kidney-derived 293T cells, and computed about 190,000 guides aimed at about 40% of human exons.[12] The other edited human and mouse cells and showed that several guides could be encoded in one array to cut several sites at once.[13]

A double-stranded break is repaired by the cell, often imprecisely, and that is how a gene gets knocked out. Variants were soon built to avoid the break. A base editor converts one target base to another without cutting both strands,[14] and prime editing searches and replaces short stretches of sequence without a break or a donor DNA.[15]

Medicine and diagnostics

In a trial reported in 2021, cells from two patients, one with β-thalassemia and one with sickle cell disease, were edited at the enhancer of the BCL11A gene and returned to them. More than a year later both patients had high levels of edited alleles and raised fetal hemoglobin, and the patient with sickle cell disease had no more vaso-occlusive episodes.[16] The therapy, Casgevy, was approved by the Food and Drug Administration on December 8, 2023 for sickle cell disease in patients aged 12 and over.[1]

Other Cas proteins have been put to work as sensors. Cas13a, which cuts RNA, and Cas12a, which cuts single-stranded DNA once it has bound its target, both go on to cut nearby reporter molecules, a property used in tests for nucleic acids.[17][18] A catalytically dead Cas9 bound at chosen sites has also been used as a marker on DNA that a nanopore can read, as a barcode.[19]

At Molecular Reality

The CV of Kent Kemmish lists work recruiting the early team at Synthego, which became a large supplier of CRISPR reagents, and running proof-of-concept directed-evolution experiments there.[20] The molecule appears in the company's games. Moleculoids has an item named CRISPR, described as "Instant kill 3 nearest enemies".[a][21] A planned arcade game on the MoleculArcade hub, Crispr Command, is described in these words: "Guide your Cas9 complex to invading viral DNA sequences. Match the PAM site, cut the target, and defend the genome!"[21]

See also

Notes

  1. ^ The item cannot drop from any monster that appears in the game, and a comment in its code says its effect is "handled in main.js", where no code handles it. See Moleculoid items.

References

  1. ^ a b "FDA approves first gene therapies to treat patients with sickle cell disease" (Press release). U.S. Food and Drug Administration. December 8, 2023.
  2. ^ Ishino, Y.; et al. (1987). "Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product". Journal of Bacteriology 169 (12): 5429-5433. doi:10.1128/jb.169.12.5429-5433.1987.
  3. ^ Ishino, Y.; Krupovic, M.; Forterre, P. (2018). "History of CRISPR-Cas from encounter with a mysterious repeated sequence to genome editing technology". Journal of Bacteriology 200 (7): e00580-17. doi:10.1128/jb.00580-17.
  4. ^ Jansen, R.; et al. (2002). "Identification of genes that are associated with DNA repeats in prokaryotes". Molecular Microbiology 43 (6): 1565-1575. doi:10.1046/j.1365-2958.2002.02839.x.
  5. ^ Mojica, F. J. M.; et al. (2005). "Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements". Journal of Molecular Evolution 60 (2): 174-182. doi:10.1007/s00239-004-0046-3.
  6. ^ Barrangou, R.; et al. (2007). "CRISPR provides acquired resistance against viruses in prokaryotes". Science 315 (5819): 1709-1712. doi:10.1126/science.1138140.
  7. ^ Garneau, J. E.; et al. (2010). "The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA". Nature 468 (7320): 67-71. doi:10.1038/nature09523.
  8. ^ Deltcheva, E.; et al. (2011). "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III". Nature 471 (7340): 602-607. doi:10.1038/nature09886.
  9. ^ Jinek, M.; et al. (2012). "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity". Science 337 (6096): 816-821. doi:10.1126/science.1225829.
  10. ^ Gasiunas, G.; et al. (2012). "Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria". Proceedings of the National Academy of Sciences 109 (39): E2579-E2586. doi:10.1073/pnas.1208507109.
  11. ^ "The Nobel Prize in Chemistry 2020". Nobel Foundation. October 7, 2020.
  12. ^ Mali, P.; et al. (2013). "RNA-guided human genome engineering via Cas9". Science 339 (6121): 823-826. doi:10.1126/science.1232033.
  13. ^ Cong, L.; et al. (2013). "Multiplex genome engineering using CRISPR/Cas systems". Science 339 (6121): 819-823. doi:10.1126/science.1231143.
  14. ^ Komor, A. C.; et al. (2016). "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage". Nature 533 (7603): 420-424. doi:10.1038/nature17946.
  15. ^ Anzalone, A. V.; et al. (2019). "Search-and-replace genome editing without double-strand breaks or donor DNA". Nature 576 (7785): 149-157. doi:10.1038/s41586-019-1711-4.
  16. ^ Frangoul, H.; et al. (2021). "CRISPR-Cas9 gene editing for sickle cell disease and β-thalassemia". New England Journal of Medicine 384 (3): 252-260. doi:10.1056/NEJMoa2031054.
  17. ^ Gootenberg, J. S.; et al. (2017). "Nucleic acid detection with CRISPR-Cas13a/C2c2". Science 356 (6336): 438-442. doi:10.1126/science.aam9321.
  18. ^ Chen, J. S.; et al. (2018). "CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity". Science 360 (6387): 436-439. doi:10.1126/science.aar6245.
  19. ^ Weckman, N. E.; et al. (2019). "Multiplexed DNA identification using site specific dCas9 barcodes and nanopore sensing". ACS Sensors 4 (8): 2065-2072. doi:10.1021/acssensors.9b00686.
  20. ^ "Kent Kemmish, curriculum vitae". Molecular Reality Corporation.
  21. ^ a b "Company records". Molecular Reality Corporation. October 2026.
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