CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) is a scientific technology used to edit genes.[1] The CRISPR acronym describes the functionality of the CRISPR gene editing process, which involves removing and replacing genetic sequences of choice. [2] The DNA of interest is found in clustered, regularly spaced sequences and is uniquely identifiable for the purpose of deleting or incorporating specific genetic information – particularly foreign DNA. Genetic palindrome sequences – which read identically from the 5’ end to the 3’ end on one DNA strand and on the complementary DNA strand from the 5’ end to the 3’ end – are distributed widely throughout the genome and play an important role in the editing process.[3] [4] Cas9 is an RNA-guided endonuclease enzyme that cleaves DNA once the genetic sequence of interest is identified.[5]

Image: DNA. Online: https://www.publicdomainpictures.net/en/view-image.php?image=31530&picture=structure-of-dna. CC0 Public Domain

Interspaced short palindromic repeats are found in both humans and bacteria. In nature, CRISPR-Cas9 (The video presented in this hyperlink which provides an excellent overview of CRISPR-Cas9) protects bacteria from infection from bacteriophages (viruses) and plasmids.[6] [7] The sheer enormity of the size of bacteriophage populations is such that bacteria employ a near constant state of surveillance from threats of infection.[8]  CRISPR builds upon its predecessor, the restriction endonuclease, in that CRISPR replaces simple cleavage of genetic sequences of interest with editing systems that possess the capacity for organism protection and learned immunity.[9] The CRISPR-Cas9 system consists of a nuclease enzyme that cleaves nucleic acids combined with two sequences of RNA – crRNA and tracrRNA. GuideRNA, or gRNA, results upon the combination of crRNA and tracrRNA.[10] [11] Once a bacterium is inoculated with a virus, spacer sequences from the viral genome are integrated into the bacterial DNA. The newly modified bacterial genetic sequence with the included genetic spacers is then transcribed into RNA. Directed by RNA (guide RNA or gRNA), the Cas9 complex targets the foreign DNA sequences.[12] [13] The CRISPR-Cas9 system then identifies, excises, and processes this foreign DNA while bolstering and imbuing perpetual bacterial immunity to the targeted viruses and plasmids.[14]

CRISPR gene editing technology has revolutionized the scientific community. CRISPR-Cas9 can accurately target and alter genes more quickly and economically than other types of genetic technology.[1] The utility of the technology is broad, globally impacting diverse fields in ever-increasing capacity on a daily basis. CRISPR-Cas9 is technologically advantageous to several other gene editing technologies in that CRISPR-Cas9 is capable of efficiently targeting many genomic loci at once.[2]

Uses of CRISPR include making plants resistant to bacteria and fungi in order to obtain higher crop yields, enhancing biofuels, engineering safer and healthier red meats, and reducing transmissibility of mosquito-borne diseases such as malaria and dengue fever.[3] [4] [5] Successful use of CRISPR has been documented in several different species, including yeast, fruit flies, mice, rats, rabbits, zebrafish, pigs, macaques, of course, humans.[6] In humans, CRISPR technology has many potential practical uses, including therapeutic – synthetic vehicles for drug delivery, drug development, and correction of mutations associated with certain diseases.[7] CRISPR has edited genetic sequences and shown clinical promise in patients with muscular dystrophy and other neuromuscular diseases.[8] [9] Therapy for several different types of cancers – including lung cancer, may be realized in the future.[10] CRISPR has also shown great promise in the treatment of hematologic diseases such as β-thalessemia and malaria.[11] HIV/AIDS and Hepatitis B are viral diseases for which ongoing CRISPR investigation has yielded promising utility.[12] [13] [14] [15] Continued research and discovery will elucidate progress in this area.

Resources for Further Reading:

1. Genome Editing with CRISPR-Cas9. McGovern Institute for Brain Research at MIT. https://www.youtube.com/watch?v=2pp17E4E-O8. Retreived 11 December 2018.
2. CRISPR/Cas-9 – Molecular Scissors Made of Enzyme and RNA. Max-Planck-Gesselschaft Institute. https://www.mpg.de/11823385/crispr-cas9.
3. CRISPR 101 eBook. Synthego. 2018. https://www.synthego.com/learn/crispr. Retrieved 17 December 2018.

Dorkina Myrick, MD, PhD, MPP, is a physician-scientist trained at the National Institutes of Health in Bethesda, Maryland.  She is currently a JD candidate at the Boston University School of Law.

References:

[1] CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats- Clustered Regularly Interspaced Short Palindromic Repeats – 9)

[2] Jeffry D Sander & J Keith Joung.“CRISPR-Cas systems for editing, regulating and targeting genomes. Nature. Nature Biotechnology 32, 347–355 (2014). Online: http://www.nature.com/nbt/journal/v32/n4/full/nbt.2842.html. Retrieved 11 December 2018.

[3] Smith, Gerald. Meeting DNA Palindromes Head to Head. Genes & Development 22:2612–2620. 2008. Online: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2751023/pdf/2612.pdf. Retrieved December 17, 2018.

[4] Lu Le, Jia, Hui, Dröge Peter, Li Jinming. The human genome-wide distribution of DNA palindromes. Functional and Integrative Genomics (2007) 7: 221. Online: https://link.springer.com/content/pdf/10.1007%2Fs10142-007-0047-6.pdf. Retrieved 17 December 2018.

[5] Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature. 2014;507(7490):62-7.

[6] Genome Editing with CRISPR-Cas9. McGovern Institute for Brain Research at MIT. https://www.youtube.com/watch?v=2pp17E4E-O8. Retrieved 11 December 2018.

[7] E.V. Orlova. How viruses infect bacteria. The EMBO Journal 28(7):797-8. May 2009. Online: https://www.researchgate.net/publication/24263323_How_viruses_infect_bacteria. Retrieved 11 December 2018.

[8] Wiedenheft B, Sternberg SH, Doudna JA. RNA-guided genetic silencing systems in bacteria and archaea. Nature. 2012;482:331–338.

[9] Danna, Kathleen and Daniel. Specific Cleavage of Simian Virus 40 DNA by Restriction Endonuclease of Hemophilus Influenzae. Proceedings of the National Academy of Sciences. USA. Vol. 68, No. 12, pp. 2913-2917, December 1971. Online: https://www.jstor.org/stable/pdf/61110.pdf?refreqid=excelsior%3A9eb40bf2f578ba9ad2efd9478541b03c. Retrieved 11 December 2018.

[10] Kato-Inui T, Takahashi G, Hsu S, Miyaoka Y. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 with improved proof-reading enhances homology-directed repair. Nucleic Acids Res. 2018; 46(9):4677-4688. Retrieved 15 December 2018.

[11] Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012; 337:816–821. Retrieved 15 December 2018.

[12] Nuñez, J. K., Lee, A. S. Y., Engelman, A. & Doudna, J. A. Nature 519, 193–198 (2015).

[13] Ledford, Heidi. CRISPR’s Mysteries. Nature. Vol. 541. pp. 280-283. 19 January 2017. Online: https://www.nature.com/polopoly_fs/1.21294!/menu/main/topColumns/topLeftColumn/pdf/541280a1.pdf. Retrieved 11 December 2018.

[14] Philippe Horvath, Rodolphe Barrangou. CRISPR/Cas, the Immune System of Bacteria and Archaea. Science. pp. 167-170. 08 January 2010. Retrieved 11 December 2018.

[15] What are genome editing and CRISPR-Cas9? Genetics Home Reference. National Library of Medicine. https://ghr.nlm.nih.gov/primer/genomicresearch/genomeediting. Retrieved 11 December 2018.

[16] F Ann Ran, Patrick D Hsu, Jason Wright, Vineeta Agarwala, David A Scott & Feng Zhang. Genome Engineering Using the CRISPR-Cas9 System. Nature Protocols volume 8, pages 2281–2308 (2013).

[17] Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014;157(6):1262-78. Online: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4343198/. Retrieved 11 December 2018.

[18] Ghorbal M, Gorman M, Macpherson CR, Martins RM, Scherf A, Lopez-Rubio JJ: Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system. Nat Biotechnol 2014, doi: 10.1038/nbt.2925. Retrieved 11 December 2018.

[19] Jaganathan D, Ramasamy K, Sellamuthu G, Jayabalan S, Venkataraman G. CRISPR for Crop Improvement: An Update Review. Frontiers in Plant Sciences. 17 July 2018

Online: https://www.frontiersin.org/articles/10.3389/fpls.2018.00985/full. Retrieved 11 December 2018.

[20] “The Age of the Red Pen.” The Economist. 22 August 2015. http://www.economist.com/news/briefing/21661799-it-now-easy-edit-genomes-plants-animals-and-humans-age-red-pen

[21] Wang E et al. Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains. Nature Biotechnology. 33, pages 661–667 (2015). Online: http://www.nature.com/nbt/journal/v33/n6/full/nbt.3235.html. Retrieved 27 December 2018.

[22] Id at Cyranoski, David.

[23] Long C, Amoasii L, Bassel-Duby R, Olson EN. Genome Editing of Monogenic Neuromuscular Diseases: A Systematic Review. JAMA Neurol.2016;73(11):1349–1355. Online: http://jamanetwork.com/journals/jamaneurology/article-abstract/2553836. Retrieved 10 December 2018.

[24] Cyranoski, David. CRISPR Gene-Editing Tested In A Person For The First Time. Nature. 15 November 2016. Online: Http://Www.Nature.Com/News/Crispr-Gene-Editing-Tested-In-A-Person-For-The-First-Time-1.20988. Retrieved 10 December 2018.

[25] Hammond A et al. A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae. Nature Biotechnology. Volume 34. Number 1. pp. 78-83. OM 1-2. January 2016. Online: http://www.nature.com/nbt/journal/v34/n1/pdf/nbt.3439.pdf. Retrieved 27 December 2018.

[26] Hultquist et al. A Cas9 Ribonucleoprotein Platform
for Functional Genetic Studies of HIV-Host Interactions in Primary Human T Cells. Cell Reports. 17, 1438–1452. October 25, 2016. Online: https://www.cell.com/action/showPdf?pii=S2211-1247%2816%2931336-5. Retrieved December 17, 2018.

[27] Khalili K, White MK, Jacobson JM. Novel AIDS therapies based on gene editing. Cell Mol Life Sci. 2017;74(13):2439-2450. Online: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5474186/pdf/nihms853462.pdf. Retrieved December 17, 2018.

[28] Li H, Sheng C, Wang S, et al. Removal of Integrated Hepatitis B Virus DNA Using CRISPR-Cas9. Front Cell Infect Microbiol. 2017;7:91. 22 March 2017. Retrieved December 27, 2018.

[29] Xing Liu, Tuidong Hao, Shuliang Chen, Deyin Guo, an Yu Chen. Inhibition of Hepatitis B virus by the CRISPR/Cas9 system via targeting the conserved regions of the viral genome. Journal of General Virology. 96, 2252-2261. 2015. Online: https://www.microbiologyresearch.org/docserver/fulltext/jgv/96/8/2252_vir000159.pdf?expires=1545962675&id=id&accname=guest&checksum=F2E004D6841CEC16172B63E0B4454A97. Retrieved December 27, 2018.