<italic toggle="yes">In Vitro</italic> CRISPR/Cas9 System for Efficient Targeted DNA Editing

ABSTRACT The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, an RNA-guided nuclease for specific genome editing in vivo, has been adopted in a wide variety of organisms. In contrast, the in vitro application of the CRISPR/Cas9 system has r...

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Autores principales: Yunkun Liu, Weixin Tao, Shishi Wen, Zhengyuan Li, Anna Yang, Zixin Deng, Yuhui Sun
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Publicado: American Society for Microbiology 2015
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spelling oai:doaj.org-article:1eed16433b004c86b10ac002d91cf6492021-11-15T15:41:23Z<italic toggle="yes">In Vitro</italic> CRISPR/Cas9 System for Efficient Targeted DNA Editing10.1128/mBio.01714-152150-7511https://doaj.org/article/1eed16433b004c86b10ac002d91cf6492015-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01714-15https://doaj.org/toc/2150-7511ABSTRACT The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, an RNA-guided nuclease for specific genome editing in vivo, has been adopted in a wide variety of organisms. In contrast, the in vitro application of the CRISPR/Cas9 system has rarely been reported. We present here a highly efficient in vitro CRISPR/Cas9-mediated editing (ICE) system that allows specific refactoring of biosynthetic gene clusters in Streptomyces bacteria and other large DNA fragments. Cleavage by Cas9 of circular pUC18 DNA was investigated here as a simple model, revealing that the 3′→5′ exonuclease activity of Cas9 generates errors with 5 to 14 nucleotides (nt) randomly missing at the editing joint. T4 DNA polymerase was then used to repair the Cas9-generated sticky ends, giving substantial improvement in editing accuracy. Plasmid pYH285 and cosmid 10A3, harboring a complete biosynthetic gene cluster for the antibiotics RK-682 and holomycin, respectively, were subjected to the ICE system to delete the rkD and homE genes in frame. Specific insertion of the ampicillin resistance gene (bla) into pYH285 was also successfully performed. These results reveal the ICE system to be a rapid, seamless, and highly efficient way to edit DNA fragments, and a powerful new tool for investigating and engineering biosynthetic gene clusters. IMPORTANCE Recent improvements in cloning strategies for biosynthetic gene clusters promise rapid advances in understanding and exploiting natural products in the environment. For manipulation of such biosynthetic gene clusters to generate valuable bioactive compounds, efficient and specific gene editing of these large DNA fragments is required. In this study, a highly efficient in vitro DNA editing system has been established. When combined with end repair using T4 DNA polymerase, Cas9 precisely and seamlessly catalyzes targeted editing, including in-frame deletion or insertion of the gene(s) of interest. This in vitro CRISPR editing (ICE) system promises a step forward in our ability to engineer biosynthetic pathways.Yunkun LiuWeixin TaoShishi WenZhengyuan LiAnna YangZixin DengYuhui SunAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 6, Iss 6 (2015)
institution DOAJ
collection DOAJ
language EN
topic Microbiology
QR1-502
spellingShingle Microbiology
QR1-502
Yunkun Liu
Weixin Tao
Shishi Wen
Zhengyuan Li
Anna Yang
Zixin Deng
Yuhui Sun
<italic toggle="yes">In Vitro</italic> CRISPR/Cas9 System for Efficient Targeted DNA Editing
description ABSTRACT The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, an RNA-guided nuclease for specific genome editing in vivo, has been adopted in a wide variety of organisms. In contrast, the in vitro application of the CRISPR/Cas9 system has rarely been reported. We present here a highly efficient in vitro CRISPR/Cas9-mediated editing (ICE) system that allows specific refactoring of biosynthetic gene clusters in Streptomyces bacteria and other large DNA fragments. Cleavage by Cas9 of circular pUC18 DNA was investigated here as a simple model, revealing that the 3′→5′ exonuclease activity of Cas9 generates errors with 5 to 14 nucleotides (nt) randomly missing at the editing joint. T4 DNA polymerase was then used to repair the Cas9-generated sticky ends, giving substantial improvement in editing accuracy. Plasmid pYH285 and cosmid 10A3, harboring a complete biosynthetic gene cluster for the antibiotics RK-682 and holomycin, respectively, were subjected to the ICE system to delete the rkD and homE genes in frame. Specific insertion of the ampicillin resistance gene (bla) into pYH285 was also successfully performed. These results reveal the ICE system to be a rapid, seamless, and highly efficient way to edit DNA fragments, and a powerful new tool for investigating and engineering biosynthetic gene clusters. IMPORTANCE Recent improvements in cloning strategies for biosynthetic gene clusters promise rapid advances in understanding and exploiting natural products in the environment. For manipulation of such biosynthetic gene clusters to generate valuable bioactive compounds, efficient and specific gene editing of these large DNA fragments is required. In this study, a highly efficient in vitro DNA editing system has been established. When combined with end repair using T4 DNA polymerase, Cas9 precisely and seamlessly catalyzes targeted editing, including in-frame deletion or insertion of the gene(s) of interest. This in vitro CRISPR editing (ICE) system promises a step forward in our ability to engineer biosynthetic pathways.
format article
author Yunkun Liu
Weixin Tao
Shishi Wen
Zhengyuan Li
Anna Yang
Zixin Deng
Yuhui Sun
author_facet Yunkun Liu
Weixin Tao
Shishi Wen
Zhengyuan Li
Anna Yang
Zixin Deng
Yuhui Sun
author_sort Yunkun Liu
title <italic toggle="yes">In Vitro</italic> CRISPR/Cas9 System for Efficient Targeted DNA Editing
title_short <italic toggle="yes">In Vitro</italic> CRISPR/Cas9 System for Efficient Targeted DNA Editing
title_full <italic toggle="yes">In Vitro</italic> CRISPR/Cas9 System for Efficient Targeted DNA Editing
title_fullStr <italic toggle="yes">In Vitro</italic> CRISPR/Cas9 System for Efficient Targeted DNA Editing
title_full_unstemmed <italic toggle="yes">In Vitro</italic> CRISPR/Cas9 System for Efficient Targeted DNA Editing
title_sort <italic toggle="yes">in vitro</italic> crispr/cas9 system for efficient targeted dna editing
publisher American Society for Microbiology
publishDate 2015
url https://doaj.org/article/1eed16433b004c86b10ac002d91cf649
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