Highly efficient multiplex editing: one-shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants
| Autor: | Karen Barthel, Johannes Stuttmann, Jessica L. Erickson, Sylvestre Marillonnet, Filiz Ferik, Rosalie Herr, Jana Ordon, Ulla Bonas, Patrick Martin, Jens Keilwagen, Carola Kretschmer, Thomas Berner |
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| Rok vydání: | 2021 |
| Předmět: |
0106 biological sciences
0301 basic medicine Mutant Arabidopsis Nicotiana benthamiana Plant Science Computational biology 01 natural sciences 03 medical and health sciences Genome editing Tobacco Genetics Arabidopsis thaliana Guide RNA Gene Gene Editing biology Cas9 fungi food and beverages Cell Biology biology.organism_classification Plants Genetically Modified 030104 developmental biology Mutation CRISPR-Cas Systems Genome Plant 010606 plant biology & botany |
| Zdroj: | The Plant journal : for cell and molecular biologyReferences. 106(1) |
| ISSN: | 1365-313X |
| Popis: | Genome editing by RNA-guided nucleases, such as SpCas9, has been used in numerous different plant species. However, to what extent multiple independent loci can be targeted simultaneously by multiplexing has not been well documented. Here, we developed a toolkit, based on a highly intron-optimized zCas9i gene, which allows assembly of nuclease constructs expressing up to 32 single guide RNAs (sgRNAs). We used this toolkit to explore the limits of multiplexing in two major model species, and report on the isolation of transgene-free octuple (8×) Nicotiana benthamiana and duodecuple (12×) Arabidopsis thaliana mutant lines in a single generation (T1 and T2 , respectively). We developed novel counter-selection markers for N. benthamiana, most importantly Sl-FAST2, comparable to the well-established Arabidopsis seed fluorescence marker, and FCY-UPP, based on the production of toxic 5-fluorouracil in the presence of a precursor. Targeting eight genes with an array of nine different sgRNAs and relying on FCY-UPP for selection of non-transgenic T1 , we identified N. benthamiana mutant lines with astonishingly high efficiencies: All analyzed plants carried mutations in all genes (approximately 112/116 target sites edited). Furthermore, we targeted 12 genes by an array of 24 sgRNAs in A. thaliana. Efficiency was significantly lower in A. thaliana, and our results indicate Cas9 availability is the limiting factor in such higher-order multiplexing applications. We identified a duodecuple mutant line by a combination of phenotypic screening and amplicon sequencing. The resources and results presented provide new perspectives for how multiplexing can be used to generate complex genotypes or to functionally interrogate groups of candidate genes. |
| Databáze: | OpenAIRE |
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