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

• Published in: The Plant journal : for cell and molecular biology Vol. 106; no. 1; pp. 8 - 22
• Main Authors: Stuttmann, Johannes, Barthel, Karen, Martin, Patrick, Ordon, Jana, Erickson, Jessica L., Herr, Rosalie, Ferik, Filiz, Kretschmer, Carola, Berner, Thomas, Keilwagen, Jens, Marillonnet, Sylvestre, Bonas, Ulla
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01-04-2021
• Subjects: 5-Fluorouracil; Arabidopsis; Arabidopsis - genetics; Arabidopsis thaliana; Arrays; Biomarkers; CRISPR-Cas Systems - genetics; CRISPR/Cas9; Editing; Fluorescence; Gene Editing; Genes; Genome, Plant - genetics; Genomes; Genotypes; Multiplexing; Mutants; Mutation; Mutation - genetics; Nicotiana - genetics; Nicotiana benthamiana; Nuclease; Plant species; Plants, Genetically Modified - genetics; RNA editing; RNA‐guided nucleases (RGNs); selection markers; technical advance; Toolkits; Arabidopsis thaliana; RNA-guided nucleases (RGNs); Nicotiana benthamiana; CRISPR/Cas9; multiplexing; technical advance; selection markers
• DOI: 10.1111/tpj.15197
• EISSN: 1365-313X
• ISSN: 0960-7412

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 (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.
Significance Statement
The limits of multiplexing in genome editing by RNA‐guided nucleases are explored, and new markers for transgene positive/negative selection are developed. This opens up new perspectives for the generation of complex genotypes by genome editing and for the use of RNA‐guided nucleases for forward genetics.