Looking to make CRISPR breaks in the genome? Well the CRISPR/Cas9 system could be the future of genome editing. The system uses the clustered regulatory interspaced short palindromic repeats (CRISPR) associated 9 (Cas9) bacterial endonuclease.
The unique aspect of this system is a short guide RNA (gRNA) is used to target the Cas9 protein to a genomic region of interest. The gRNA has a hairpin component that is recognized by Cas9 and a variable region that binds DNA and confers specify. The protein and RNA components can be expressed from a vector after being transfected into the cell along with the DNA to be inserted into the break. It is highly efficient, cheap, and easy (Wei el al., 2013). Only the RNA needs to the modified to target regions of interest, so multiple RNAs can be used in a single experiment to target multiple regions.
There is however a downside, the gRNA has a shorter recognition sequence than ZFNs or TALENs meaning it has the potential for more off-target effects. Indeed, these effects have been shown to occur in human cells (Fu et al., 2013). This effect may be able to be overcome as research into the precise mechanism of action of Cas9 is currently a hot topic (Jinek et al., 2014; Sternberg et al., 2014). The Cas9 system has been used to upregulate and downregulate endogenous genes (Gilbert et al., 2013).
CRISPR/Cas9 Additional Reading
This perspective article discusses the origins and development of the CRISPR/Cas9 system as wells some of its applications so far.
This paper describes the Cas9 system in detail, focusing on the endogenous function of Cas9 in bacteria, function of the system in genome editing, potential applications, and work still to be done.
- Fu, Y., Foden, J.A., Khayter, C., Maeder, M.L., Reyon, D., Joung, J.K., and Sander, J.D. (2013). High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31, 822-826.
- Gilbert, L.A., Larson, M.H., Morsut, L., Liu, Z., Brar, G.A., Torres, S.E., Stern-Ginossar, N., Brandman, O., Whitehead, E.H., Doudna, J.A., et al. (2013). CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442-451.
- Jinek, M., Jiang, F., Taylor, D.W., Sternberg, S.H., Kaya, E., Ma, E., Anders, C., Hauer, M., Zhou, K., Lin, S., et al. (2014). Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation. Science
- Sternberg, S.H., Redding, S., Jinek, M., Greene, E.C., and Doudna, J.A. (2014). DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507, 62-67.
- Wei, C., Liu, J., Yu, Z., Zhang, B., Gao, G., and Jiao, R. (2013). TALEN or Cas9 – rapid, efficient and specific choices for genome modifications. J. Genet. Genomics 40, 281-289.