CRISPR-Cas9: the new golden child of genetic engineering – RCSIsmj staff writer Vincent Healy
Lauded as the biggest game-changer since PCR, the humble enzyme Cas9 (CRISPR-associated nuclease 9) is keeping molecular biologists awake at night.
CRISPR-Cas9 is the latest genetic engineering technique using site-directed cleaving enzymes as ‘molecular scissors’. Cas9 is an RNA-guided DNA endonuclease, while CRISPRs are viral nucleic acid sequences which have been incorporated into bacterial genomes. Bacteria use these sequences to recognise and cut matching viral DNA, thus avoiding infection.1-5
Inserting customized CRISPR-Cas9 elements into eukaryotic cells allows genome alteration at any position.1-5 Gene insertion could correct gene function in genetic disease or enhance favourable crop attributes in agriculture, while gene knock-out is key to disease model research such as in cancer.1-5 Another technique, gene drive, rapidly spreads mutations, which could be used to cripple mosquito populations and impact malaria rates.6, 7
RESEARCH & CHALLENGES
CRISPR has significantly increased the accessibility and usability of endonuclease editing platforms, being cheap, quick and easily modifiable.2,8 A competitor, Zinc Finger Nucleases, costs in excess of $5,000 to order; CRISPR-Cas9 can be as low as $30.8
In April 2015, a Chinese paper reported having used CRISPR-Cas9 to edit human embryos – specifically the gene responsible for β-thalassemia. Attempting to head off ethical opposition, researchers used nonviable triploid embryos to circumvent issues of heritability.10, 11 However, only a fraction were correctly spliced while less actually contained the replacement genetic material.11Similarly disappointing was an MIT study – in murine adult liver cells – finding their intended mutation had only occurred in 0.4% of the cell population.8
Low efficacy, high levels of off-target mutations and disruptive cleavage have led the embryo researchers to redirect their efforts, calling for improving fidelity and specificity of CRISPR-Cas9 platforms in other models before further embryo research.11
Conversely, in a significant step for xenotransplantation, Harvard researchers announced in October 2015 successful CRISPR-mediated modification of record numbers of porcine genes towards enabling disease- and rejection-free pig-to-human organ transplant.12
With the Chinese embryo CRISPR platforms already considered outdated, CRISPR is evolving rapidly.10
From 2013 to 2014, US National Institutes of Health CRISPR funding jumped from $10 million to above $80 million.8 UK scientists recently submitted for regulatory approval to edit human embryonic DNA, while sources report at least four additional Chinese groups continuing human embryo research.10
CRISPR might yet grow up to eclipse PCR as ‘the’ game-changer in molecular biology … watch this space.
1. Sternberg SH, Doudna JA. Expanding the Biologist’s Toolkit with CRISPR-Cas9. Molecular cell. 2015;58(4):568-74.
2. Hartenian E, Doench JG. Genetic screens and functional genomics using CRISPR/Cas9 technology. The FEBS journal. 2015;282(8):1383-93.
3. Zhang F, Wen Y, Guo X. CRISPR/Cas9 for genome editing: progress, implications and challenges. Human molecular genetics. 2014;23(R1):R40-6.
4. Xiao-Jie L, Hui-Ying X, Zun-Ping K, Jin-Lian C, Li-Juan J. CRISPR-Cas9: a new and promising player in gene therapy. Journal of medical genetics. 2015;52(5):289-96.
5. Cong L, Zhang F. Genome engineering using CRISPR-Cas9 system. Methods in molecular biology (Clifton, NJ). 2015;1239:197-217.
6. Esvelt KM, Smidler AL, Catteruccia F, Church GM. Concerning RNA-guided gene drives for the alteration of wild populations. eLife. 2014;3.
7. Webber BL, Raghu S, Edwards OR. Opinion: Is CRISPR-based gene drive a biocontrol silver bullet or global conservation threat? Proc Natl Acad Sci U S A. 2015;112(34):10565-7.
8. Leiford H. CRISPR, the disruptor. Nature News & Comment. 2015 08 June 2015.
9. D. C. Super-muscly pigs created by small genetic tweak. Nature News & Comment. 2015.
10. Cyranoski D. RS. Embryo editing sparks epic debate. Nature News & Comment. 2015 29 April 2015.
11. Liang P, Xu Y, Zhang X, Ding C, Huang R, Zhang Z, et al. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell. 2015;6(5):363-72.
12. S. R. Gene-editing record smashed in pigs. Nature News & Comment. 2015 06 October 2015.
Image adapted by permission from Macmillan Publishers Ltd: Nature (Charpentier et al.,Biotechnology:Rewriting a genome), copyright (2013)