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

作　　者：Ramona Grützner Patrick Martin Claudia Horn Samuel Mortensen Erin J.Cram Carolyn W.T.Lee-Parsons Johannes Stuttmann Sylvestre Marillonnet 

机构地区：[1]Department of Cell and Metabolic Biology,Leibniz Institute of Plant Biochemistry,Weinberg 3,06120 Halle(Saale),Germany [2]Institute for Biology,Department of Plant Genetics,Martin Luther University Halle-Wittenberg,Weinbergweg 10,06120 Halle(Saale),Germany [3]Department of Biology,Northeastern University,Boston,MA,USA [4]Department of Chemistry and Chemical Biology,Northeastern University,Boston,MA,USA [5]Department of Chemical Engineering,Northeastern University,Boston,MA,USA

出　　处：《Plant Communications》2021年第2期63-77,共15页植物通讯（英文）

基　　金：supported by core funding of the IPB;supported by grant STU642-1/1(DFG/GRC,Deutsche Forschungsgemeinschaft);supported by the National Science Foundation MCB award 1516371 to C.L.-P.and E.C.

摘　　要：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 interest.In plants,site-targeted mutations are usually obtained by the stable transformation of a Cas9 expression construct into the plant genome.The 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 itself.To 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 introns.We found that the Cas9 gene codon usage had some effect on its activity and that two NLSs worked better than one.However,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 constructs.None 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 phenotypes.The 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 

分 类 号：Q943.2[生物学—植物学]