Genome editing using CRISPR/ Cas9 system: a practical guide

Over the past few years, the CRISPR/Cas techniques have become a revolution in genome editing. Since the original paper on CRIPSR/Cas9 genome editing, researches have proposed numerous modifications of the key components of the CRISPR/Cas9 system to make it extremely efficient. Nowadays, CRISPR/Cas ...

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Main Authors: A. G. Menzorov, V. A. Lukyanchikova, A. N. Korablev, I. A. Serova, V. S. Fishman
Format: Article
Language:English
Published: Siberian Branch of the Russian Academy of Sciences, Federal Research Center Institute of Cytology and Genetics, The Vavilov Society of Geneticists and Breeders 2017-02-01
Series:Вавиловский журнал генетики и селекции
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Online Access:https://vavilov.elpub.ru/jour/article/view/874
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author A. G. Menzorov
V. A. Lukyanchikova
A. N. Korablev
I. A. Serova
V. S. Fishman
author_facet A. G. Menzorov
V. A. Lukyanchikova
A. N. Korablev
I. A. Serova
V. S. Fishman
author_sort A. G. Menzorov
collection DOAJ
description Over the past few years, the CRISPR/Cas techniques have become a revolution in genome editing. Since the original paper on CRIPSR/Cas9 genome editing, researches have proposed numerous modifications of the key components of the CRISPR/Cas9 system to make it extremely efficient. Nowadays, CRISPR/Cas systems can be used not only to modify genomes, but also to control expression levels of defined genes, visualize loci of interest in the space of living cell nuclei, change methylation status of mammalian CpG sites, and to serve many other purposes. Due to an extremely high efficacy and ease of usage, the CRISPR/ Cas system has been employed in a large number of studies in various areas of biology and biotechnology. We have recently published a review describing various CRISPR/Cas systems, mechanisms of their functioning, and applications of the techniques in details. Despite the broad range of potential applications of CRISPR/Cas systems, they are mostly used for genome editing. And, however simple the system may be, there is a number of potential pitfalls on the way towards its use in CRISPR/Cas- naïve laboratory settings. In this article, we describe protocols of CRISPR/Cas9 system generation. We start with a short description of theoretical aspects underlying Cas9-mediated genome editing. Next, we describe a step-by-step protocol of guide RNA vector design and assembly, and several ways of qualitative and quantitative evaluations of the system. Finally, we report protocols of genome editing for modification of embryonic stem cells and zygotes.
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publisher Siberian Branch of the Russian Academy of Sciences, Federal Research Center Institute of Cytology and Genetics, The Vavilov Society of Geneticists and Breeders
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series Вавиловский журнал генетики и селекции
spelling doaj-art-315cfee4dc2146179b274d5926873ee72025-02-01T09:58:04ZengSiberian Branch of the Russian Academy of Sciences, Federal Research Center Institute of Cytology and Genetics, The Vavilov Society of Geneticists and BreedersВавиловский журнал генетики и селекции2500-32592017-02-0120693094410.18699/VJ16.214561Genome editing using CRISPR/ Cas9 system: a practical guideA. G. Menzorov0V. A. Lukyanchikova1A. N. Korablev2I. A. Serova3V. S. Fishman4Institute of Cytology and Genetics SB RAS Novosibirsk State UniversityInstitute of Cytology and Genetics SB RASInstitute of Cytology and Genetics SB RASInstitute of Cytology and Genetics SB RASInstitute of Cytology and Genetics SB RAS Novosibirsk State UniversityOver the past few years, the CRISPR/Cas techniques have become a revolution in genome editing. Since the original paper on CRIPSR/Cas9 genome editing, researches have proposed numerous modifications of the key components of the CRISPR/Cas9 system to make it extremely efficient. Nowadays, CRISPR/Cas systems can be used not only to modify genomes, but also to control expression levels of defined genes, visualize loci of interest in the space of living cell nuclei, change methylation status of mammalian CpG sites, and to serve many other purposes. Due to an extremely high efficacy and ease of usage, the CRISPR/ Cas system has been employed in a large number of studies in various areas of biology and biotechnology. We have recently published a review describing various CRISPR/Cas systems, mechanisms of their functioning, and applications of the techniques in details. Despite the broad range of potential applications of CRISPR/Cas systems, they are mostly used for genome editing. And, however simple the system may be, there is a number of potential pitfalls on the way towards its use in CRISPR/Cas- naïve laboratory settings. In this article, we describe protocols of CRISPR/Cas9 system generation. We start with a short description of theoretical aspects underlying Cas9-mediated genome editing. Next, we describe a step-by-step protocol of guide RNA vector design and assembly, and several ways of qualitative and quantitative evaluations of the system. Finally, we report protocols of genome editing for modification of embryonic stem cells and zygotes.https://vavilov.elpub.ru/jour/article/view/874genome editingcrispr/cas9embryonic stem cells.
spellingShingle A. G. Menzorov
V. A. Lukyanchikova
A. N. Korablev
I. A. Serova
V. S. Fishman
Genome editing using CRISPR/ Cas9 system: a practical guide
Вавиловский журнал генетики и селекции
genome editing
crispr/cas9
embryonic stem cells.
title Genome editing using CRISPR/ Cas9 system: a practical guide
title_full Genome editing using CRISPR/ Cas9 system: a practical guide
title_fullStr Genome editing using CRISPR/ Cas9 system: a practical guide
title_full_unstemmed Genome editing using CRISPR/ Cas9 system: a practical guide
title_short Genome editing using CRISPR/ Cas9 system: a practical guide
title_sort genome editing using crispr cas9 system a practical guide
topic genome editing
crispr/cas9
embryonic stem cells.
url https://vavilov.elpub.ru/jour/article/view/874
work_keys_str_mv AT agmenzorov genomeeditingusingcrisprcas9systemapracticalguide
AT valukyanchikova genomeeditingusingcrisprcas9systemapracticalguide
AT ankorablev genomeeditingusingcrisprcas9systemapracticalguide
AT iaserova genomeeditingusingcrisprcas9systemapracticalguide
AT vsfishman genomeeditingusingcrisprcas9systemapracticalguide