Highly efficient multiplex editing: one‐shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants. (25th March 2021)

• Record Type: Journal Article
• 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
• Is Part Of: Plant journal. Volume 106:Number 1(2021)
• Page Start: 8
• Page End: 22
• Publication Date: 2021-03-25
• Subjects: CRISPR/Cas9 -- RNA‐guided nucleases (RGNs) -- multiplexing -- selection markers -- Arabidopsis thaliana -- Nicotiana benthamiana -- technical advance; Plant molecular biology -- Periodicals; Plant cells and tissues -- Periodicals; Botany -- Periodicals; 580
• DOI: 10.1111/tpj.15197
• Languages: English
• ISSNs: 0960-7412

Abstract

SUMMARY: Genome editing by RNA‐guided nucleases, such as Sp Cas9, 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.