Highly efficient multiplex editing: one-shot generation of 8x Nicotiana benthamiana and 12x Arabidopsis mutants

Keywords: CRISPR/Cas9; RNA-guided nucleases (RGNs); multiplexing; selection markers; Arabidopsis thaliana; Nicotiana benthamiana; technical advance SUMMARY Genome editing by RNA-guided nucleases, such as SpCas9, has been used in numerous different plant species. However, to what extent multiple inde...

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Published in:	The Plant journal : for cell and molecular biology 2021-04, Vol.106 (1), p.8
Main Authors:	Martin, Patrick, Erickson, Jessica L, Keilwagen, Jens, Kretschmer, Carola, Marillonnet, Sylvestre, Bonas, Ulla, Stuttmann, Johannes, Ordon, Jana, Berner, Thomas, Barthel, Karen, Herr, Rosalie, Ferik, Filiz
Format:	Article
Language:	eng
Subjects:	Analysis Arabidopsis thaliana DNA binding proteins Genetic engineering Nucleases RNA
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Summary:	Keywords: CRISPR/Cas9; RNA-guided nucleases (RGNs); multiplexing; selection markers; Arabidopsis thaliana; Nicotiana benthamiana; technical advance SUMMARY 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 (8x) Nicotiana benthamiana and duodecuple (12x) Arabidopsis thaliana mutant lines in a single generation (T.sub.1 and T.sub.2, 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 T.sub.1, 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. Article Note: Linked article: This paper is the subject of a Research Highlight article. To view this Research Highlight article visit CAPTION(S): Figure S1. Functional verification of pDGE nuclease vectors and U6/U3 promoters. Figure S2. Efficiency of Agrobacterium-mediated transformation of Nicotiana benthamiana using pDGE vectors. Figure S3. Transgene counter-selection in N. benthamiana by seed fluorescence and inducible cell death. Figure S4. Toxicity of 5-FC to Arabidopsis plants on quartz sand plates. Figure S5. Toxicity of 5-FC to N. benthamiana on quartz s
ISSN:	0960-7412 1365-313X