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使用单载体CRISPR/Cas9的基因组编辑

Genomic Edition of Using One-vector CRISPR/Cas9.

作者信息

Muñoz-Fernández Gloria, Jiménez Alberto, Revuelta José Luis

机构信息

Metabolic Engineering Group, Department of Microbiology and Genetics, University of Salamanca, Campus Miguel de Unamuno, E-37007 Salamanca, Spain.

出版信息

Bio Protoc. 2020 Jun 20;10(12):e3660. doi: 10.21769/BioProtoc.3660.

Abstract

The CRISPR/Cas9 system is a novel genetic tool which allows the precise manipulation of virtually any genomic sequence. In this protocol, we use a specific CRISPR/Cas9 system for the manipulation of . The filamentous fungus is currently used for the industrial production of riboflavin (vitamina B2). In addition, produces other high-value compounds such as folic acid, nucleosides and biolipids. A large molecular toolbox is available for the genomic manipulation of this fungus including gene targeting methods, rapid assembly of heterologous expression modules and, recently, a one-vector CRISPR/Cas9 editing system adapted for that allows marker-free engineering strategies to be implemented. The CRISPR/Cas9 system comprises an RNA guided DNA endonuclease (Cas9) and a guide RNA (gRNA), which is complementary to the genomic target region. The Cas9 nuclease requires a 5'-NGG-3' trinucleotide, called protospacer adjacent motif (PAM), to generate a double-strand break (DSB) in the genomic target, which can be repaired with a synthetic mutagenic donor DNA (dDNA) by homologous recombination (HR), thus introducing a specific designed mutation. The CRISPR/Cas9 system adapted for largely facilitates the genomic edition of this industrial fungus.

摘要

CRISPR/Cas9系统是一种新型基因工具,可对几乎任何基因组序列进行精确操作。在本方案中,我们使用特定的CRISPR/Cas9系统来操作……丝状真菌目前用于工业生产核黄素(维生素B2)。此外,……还能产生其他高价值化合物,如叶酸、核苷和生物脂质。有一个大型分子工具箱可用于该真菌的基因组操作,包括基因靶向方法、异源表达模块的快速组装,以及最近适用于……的单载体CRISPR/Cas9编辑系统,该系统允许实施无标记工程策略。CRISPR/Cas9系统由RNA引导的DNA内切酶(Cas9)和与基因组靶区域互补的引导RNA(gRNA)组成。Cas9核酸酶需要一个5'-NGG-3'三核苷酸,称为原间隔相邻基序(PAM),以在基因组靶标中产生双链断裂(DSB),该双链断裂可通过同源重组(HR)用合成诱变供体DNA(dDNA)修复,从而引入特定设计的突变。适用于……的CRISPR/Cas9系统极大地促进了这种工业真菌的基因组编辑。

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本文引用的文献

1
One-vector CRISPR/Cas9 genome engineering of the industrial fungus Ashbya gossypii.
Microb Biotechnol. 2019 Nov;12(6):1293-1301. doi: 10.1111/1751-7915.13425. Epub 2019 May 5.
2
Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae.
Metab Eng. 2015 Mar;28:213-222. doi: 10.1016/j.ymben.2015.01.008. Epub 2015 Jan 28.
3
Homology-integrated CRISPR-Cas (HI-CRISPR) system for one-step multigene disruption in Saccharomyces cerevisiae.
ACS Synth Biol. 2015 May 15;4(5):585-94. doi: 10.1021/sb500255k. Epub 2014 Sep 19.
4
Genome engineering using the CRISPR-Cas9 system.
Nat Protoc. 2013 Nov;8(11):2281-2308. doi: 10.1038/nprot.2013.143. Epub 2013 Oct 24.
5
High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity.
Nat Biotechnol. 2013 Sep;31(9):839-43. doi: 10.1038/nbt.2673. Epub 2013 Aug 11.
6
Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems.
Nucleic Acids Res. 2013 Apr;41(7):4336-43. doi: 10.1093/nar/gkt135. Epub 2013 Mar 4.
7
A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.
Science. 2012 Aug 17;337(6096):816-21. doi: 10.1126/science.1225829. Epub 2012 Jun 28.
8
CRISPR/Cas, the immune system of bacteria and archaea.
Science. 2010 Jan 8;327(5962):167-70. doi: 10.1126/science.1179555.
9
Metabolic engineering of the purine pathway for riboflavin production in Ashbya gossypii.
Appl Environ Microbiol. 2005 Oct;71(10):5743-51. doi: 10.1128/AEM.71.10.5743-5751.2005.

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