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

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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