Methods for studying genetic modifications


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Abstract

Objects and modern methods of genome editing are considered. The immune system of prokaryote and their protective mechanisms that prevent the purposeful editing of the genome for the benefit of the researcher is characterized. This mechanism in prokaryotes are cluster regulatory interspatial short palindrome repetitions. The number of such repetitions varies from object to object, which ultimately makes it impossible to get the perfect standard model. Three types of such systems that have their own mechanism for generating proteins have now been identified. The proteins, which are now most commonly used to edit the genome and identify areas of proto-special adjacent motifs, are described. Detailed characteristics of the organization of the immune system prokaryote and phases of its activity are given. Three types of short-palin re-recurrence systems have now been identified, and the teams are being identified as cluster regulatory interspatial short palindrome repetitions-Cas9. Each system uses its own mechanism to generate proteins that catalyze the fission of nucleic acids. The type II cluster regulatory interspatial short palindrome repetitions system is most commonly used, better adapted to edit the genome because of its simplicity. It has been established that the cluster regulatory interspatial short palindrome repetitions-Cas9 system can be used for point editing of the genome and in eukaryotes. This is done either through non-homological annexation of the end, or by homologically directed reparation. A promising variant of genetic modeling is the use of the enzyme-endonuclease Cpf1, which is the effector protein of the cluster regulatory interspatial short palindrome repetitions-Cas V type systems. Cpf1 is smaller than the enzyme protein Cas9 and for the system to function only require specers of ribonucleic acid, without additional ribonucleic acid. Unlike Cas9, which cuts both chains of deoxyribonucleic acid in the same place, Cpf1 generates an incision, creating «ticky» ends that can be used to insert interesting sequences by complementing and ligation. It is likely that the system using the enzyme-endonuclease Cpf1 will be more convenient than the system where the protein is used – Cas9, as the range of editing of the controlled genome of ribonucleic acid is expanded to make the necessary edits.

About the authors

A. V. Moskalev

Military medical academy of S.M. Kirov

Author for correspondence.
Email: vmeda-nio@mil.ru
Russian Federation, Saint Petersburg

B. Yu. Gumilevskiy

Military medical academy of S.M. Kirov

Email: vmeda-nio@mil.ru
Russian Federation, Saint Petersburg

V. Ya. Apchel

Military medical academy of S.M. Kirov;

Email: vmeda-nio@mil.ru
Russian Federation, Saint Petersburg

V. N. Tsygan

Military medical academy of S.M. Kirov

Email: vmeda-nio@mil.ru
Russian Federation, Saint Petersburg

References

Supplementary files

Supplementary Files
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2. Fig. 1. Introduction of foreign vectors into the CRISPR locus

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3. Fig. 2. Editing the genome with the S. pyogenes Cas9 protein

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4. Fig. 3. Cleavage of DNA by different Cas9 domains

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5. Fig. 4. The role of the CRISPR-dCas9 system in gene repression and activation

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6. Fig. 5. Creation of transgenic mice from ESC: a - cells are modified by introducing recombinant clones selected and tested. They are injected into the blastocysts of the uterus of receptor mice; b - mechanism of positive and negative selection (neo - neomycin-resistant genes. TK - thymidine kinase gene); c - one of the methods of using CRISPR-Cas9 for transgenic modification of zygotes. This is where the components are injected into the pronuclei

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Copyright (c) 2020 Moskalev A.V., Gumilevskiy B.Y., Apchel V.Y., Tsygan V.N.

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This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

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