CRISPR/Cas9

Developers

Jennifer A. Doudna, J. Huang, Z. Feng, et al.

Description of the technology

Systems of genome editing existed before today (transgenesis using homological recombination in mice, as well as use of viral vectors) do not allow to make pointwise changes in a strictly defined locus of the genome.

Molecular system CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 is an up-to-date technology for high-precision genome editing. This system is comparatively simple to construct and works with high effectiveness in human, animal and plant cells.

CRISPR/Cas9 system allows to make a double-strand break in the target region of the genome. Components of the CRISPR/Cas9 system are non-coding RNAs and Cas proteins (CRISPR-associated). The CRISPR/Cas9 system recognizes the target site by means of interaction of non-coding RNA — on the one hand, and DNA of target sites — on the other hand. This is accompanied with formation of the complex of non-coding RNA and Cas proteins which exhibit helicase and nuclease activities.

The CRISPR/Cas9 system is created on the basis of bacterial CRISPR/Cas system which is properly a unique mechanism protecting microorganisms from foreign DNA (DNA of viruses and phages) penetration and acting, together with the restriction-modification system, as a limiter of horizontal transfer of genetic information.

When work with the CRISPR/Cas9 system, careful selection of sites where specific double-strand break will be made is very important. The necessity to carry out the preparatory bioinformatical analysis is linked to the possibility of nontarget effects — making nonspecific double-strand breaks in the genome. Nowadays, on-line software is accessible, which was developed by various teams. It is intended for selection of prospective sites for the CRISPR/Cas9 system, as well as for detection probable nontarget effects.

This technology is at the preclinical trial stage.

Practical application

The CRISPR/Cas9 system allows to solve difficult tasks, including creation and investigation on the disease models on the basis of cultivated human pluripotent cells. For example, development of panels of human isogenic pluripotent stem cells will implement modelling of hereditary and multifactorial diseases, screening of large drug libraries, as well as searching for new mutations involved in the pathological process. It seems particularly relevant to study severe neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and various muscle atrophies.

Moreover, CRISPR/Cas9-based methods can be effectively used for the genome editing of cultivated stem cells. Particularly, the use of genome editing systems allows to correct point mutations in the cells obtained from patients. With this approach, so-called organoids could be produced. They are functional multicellular masses with mended genome and autological in regard to donor cells. Such organoids could be placed back into the patient’s organism. Without doubts, this technology opens great prospects for the cell therapy of human diseases.

The CRISPR/Cas9 system with catalytically ineffective dCas9 can be used as a modular platform which binds to the specified nucleotide sequence and attracts protein factors there, making it possible to use this system as the main method for precise regulation of gene expression in human cells.

Laboratories

Lawrence Berkeley National Laboratory, Berkeley, California (USA)
Guangdong Province Key Laboratory of Reproductive Medicin, Sun Yat-sen University, Guangzhou, (China)
Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, Gunma (Japan)

Links

http://cyberleninka.ru/article/n/sistemy-redaktirovaniya-genomov-talen-i-crispr-cas-instrumenty-otkrytiy
http://kot.sh/statya/208/otredaktirovat-cheloveka
https://ru.wikipedia.org/wiki/CRISPR

Publications

• Jinek, Martin, et al. «A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Science 337.6096 (2012): 816–821.
• Liang, Puping, et al. «CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes." Protein & cell 6 (2015): 363–372.
• Polcz, Sarah, and Anna Lewis. «Crispr-Cas9 and the Non-Germline Non-Controversy." Journal of Law and the Biosciences, Forthcoming (2015).
• Slaymaker, Ian M., et al. «Rationally engineered Cas9 nucleases with improved specificity." Science 351.6268 (2016): 84–88.
• Feng, Zhengyan, et al. «Efficient genome editing in plants using a CRISPR/Cas system." Cell research 23.10 (2013): 1229.
• Horii, Takuro, et al. «Generation of an ICF syndrome model by efficient genome editing of human induced pluripotent stem cells using the CRISPR system." International journal of molecular sciences 14.10 (2013): 19774–19781.