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targeted mutagenesis:A sniper-like diversity generator in microbial engineering

Synthetic and Systems Biotechnology 2017年第2期2卷 75-86页

作者： Xiang Zheng Xin-Hui Xing Chong Zhang Key Laboratory for Industrial Biocatalysis Ministry of EducationInstitute of Biochemical EngineeringDepartment of Chemical EngineeringCenter for Synthetic&Systems BiologyTsinghua UniversityBeijing 100084China

Mutations,serving as the raw materials of evolution,have been extensively utilized to increase the chances of engineering molecules or microbes with tailor-made *** and targeted mutagenesis are two main methods of obtaining various mutations,distinguished by the range of action they can *** the former one stresses the mining of novel genetic loci within the whole genomic background,targeted mutagenesis performs in a more straightforward manner,bringing evolutionary escape and error catastrophe under *** this review,we classify the existing techniques of targeted mutagenesis into two categories in terms of whether the diversity is generated in vitro or in vivo,and briefly introduce the mechanisms and applications of them *** inherent connections and development trends of the two classes are also discussed to provide an insight into the next generation evolution research.

关键词： Mutations targeted mutagenesis Evolution Synthetic biology

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Efficiency and Inheritance of targeted mutagenesis in Maize Using CRISPR-Cas9

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Journal of Genetics and Genomics 2016年第1期43卷 25-36页

作者： Jinjie Zhu Ning Song Silong Sun Weilong Yang Haiming Zhao Weibin Song Jinsheng Lai State Key Laboratory of Agrobiotechnology and National Maize Improvement Center Department of Plant Genetics and Breeding China Agricultural University

CRISPR/Cas （clustered regularly interspaced short palindromic repeats/CR1SPR-associated proteins） is an adaptive immune system in bacteria and archaea to defend against invasion from foreign DNA fragments. Recently, it has been developed as a powerful targeted genome editing tool for a wide variety of species. However, its application in maize has only been tested with transiently expressed somatic cells or with a limited number of stable transgenic To plants. The exact efficiency and specificity of the CRISPR/Cas system in the highly complex maize genome has not been documented yet. Here we report an extensive study of the well-studied type II CRISPR-Cas9 system for targeted genome editing in maize, with the codon-optimized Cas9 protein and the short non-coding guide RNA generated through a functional maize U6 snRNA promoter. targeted gene mutagenesis was detected for 90 loci by maize protoplast assay, with an average cleavage efficiency of 10.67 %. Stable knockout transformants for maize phytoene synthase gene （PSYI） were obtained. Mutations occurred in germ ceils can be stably inherited to the next generation. Moreover, no off-target effect was detected at the computationally predicted putative off-target loci. No significant difference between the transcriptomes of the Cas9 expressed and non-expressed lines was detected. Our results confirmed that the CRISPR-Cas9 could be successfully applied as a robust targeted genome editing system in maize.

关键词： CRISPR-Cas9 targeted mutagenesis Heritability Maize

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CRISPR/Cas9 and Genome Editing in Drosophila

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Journal of Genetics and Genomics 2014年第1期41卷 7-19页

作者： Andrew R.Bassett Ji-Long Liu MRC Functional Genomics Unit University of Oxford Department of Physiology Anatomy and Genetics

Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Traditional techniques for generating genetic mutations in most organisms have relied on selection from large pools of randomly induced mutations for those of particular interest, or time-consuming gene targeting by homologous recombination. Drosophila melanogaster has always been at the forefront of genetic analysis, and application of these new genome editing techniques to this organism will revolutionise our approach to performing analysis of gene function in the future. We discuss the recent techniques that apply the CRISPR/Cas9 system to Drosophila, highlight potential uses for this technology and speculate upon the future of genome engineering in this model organism.

关键词： Drosophila melanogaster CRISPR Cas9 Genome engineering targeted mutagenesis

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High-efficiencygenomeediting in plants mediated by a Cas9 gene containing multiple introns

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Plant Communications 2021年第2期2卷 63-77页

作者： Ramona Grützner Patrick Martin Claudia Horn Samuel Mortensen Erin J.Cram Carolyn W.T.Lee-Parsons Johannes Stuttmann Sylvestre Marillonnet Department of Cell and Metabolic Biology Leibniz Institute of Plant BiochemistryWeinberg 306120 Halle(Saale)Germany Institute for Biology Department of Plant GeneticsMartin Luther University Halle-WittenbergWeinbergweg 1006120 Halle(Saale)Germany Department of Biology Northeastern UniversityBostonMAUSA Department of Chemistry and Chemical Biology Northeastern UniversityBostonMAUSA Department of Chemical Engineering Northeastern UniversityBostonMAUSA

The recent discovery of the mode of action of the CRISPR/Cas9 systemhas provided biologists with a useful tool for generating site-specific mutations in genes of *** plants,site-targeted mutations are usually obtained by the stable transformation of a Cas9 expression construct into the plant *** efficiency of introducing mutations in genes of interest can vary considerably depending on the specific features of the constructs,including the source and nature of the promoters and terminators used for the expression of the Cas9 gene and the guide RNA,and the sequence of the Cas9 nuclease *** optimize the efficiency of the Cas9 nuclease in generating mutations in target genes in Arabidopsis thaliana,we investigated several features of its nucleotide and/or amino acid sequence,including the codon usage,the number of nuclear localization signals(NLSs),and the presence or absence of *** found that the Cas9 gene codon usage had some effect on its activity and that two NLSs worked better than ***,the highest efficiency of the constructs was achieved by the addition of 13 introns into the Cas9 coding sequence,which dramatically improved the editing efficiency of the *** of the primary transformants obtained with a Cas9 gene lacking introns displayed a knockout mutant phenotype,whereas between 70%and 100%of the primary transformants generated with the intronized Cas9 gene displayed mutant *** intronized Cas9 gene was also found to be effective in other plants such as Nicotiana benthamiana and Catharanthus roseus.

关键词： CRISPR Cas9 targeted mutagenesis gene targeting

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