One-shot generation of duodecuple (12x) mutant Arabidopsis: Highly efficient routine editing in model species

Affiliation
Martin Luther University, Institute for Biology, Department of Plant Genetics, Germany
Barthel, Karen;
Affiliation
Martin Luther University, Institute for Biology, Department of Plant Genetics, Germany
Martin, Patrick;
Affiliation
Martin Luther University, Institute for Biology, Department of Plant Genetics, Germany
Ordon, Jana;
Affiliation
Martin Luther University, Institute for Biology, Department of Plant Genetics, Germany
Erickson, Jessica L.;
Affiliation
Martin Luther University, Institute for Biology, Department of Plant Genetics, Germany
Gantner, Johannes;
Affiliation
Martin Luther University, Institute for Biology, Department of Plant Genetics, Germany
Herr, Rosalie;
Affiliation
Martin Luther University, Institute for Biology, Department of Plant Genetics, Germany
Kretschmer, Carola;
GND
1172920559
Affiliation
Julius Kühn-Institute (JKI), Institute for Biosafety in Plant Biotechnology, Germany
Berner, Thomas;
GND
1013858662
Affiliation
Julius Kühn-Institute (JKI), Institute for Biosafety in Plant Biotechnology, Germany
Keilwagen, Jens;
Affiliation
Leibniz Institute of Plant Biochemistry, Department of Cell and Metabolic Biology, Germany
Marillonnet, Sylvestre;
ORCID
0000-0002-6207-094X
Affiliation
Martin Luther University, Institute for Biology, Department of Plant Genetics, Germany
Stuttmann, Johannes

Genome editing by RNA-guided nucleases in model species is still hampered by low efficiencies, and isolation of transgene-free individuals often requires tedious PCR screening. Here, we present a toolkit that mitigates these drawbacks for Nicotiana benthamiana and Arabidopsis thaliana . The toolkit is based on an intron-optimized Sp Cas9-coding gene (zCas9i), which conveys dramatically enhanced editing efficiencies. The zCas9i gene is combined with remaining components of the genome editing system in recipient vectors, which lack only the user-defined guide RNA transcriptional units. Up to 32 guide RNA transcriptional units can be introduced to these recipients by a simple and PCR-free cloning strategy, with the choice of three different RNA polymerase III promoters for guide RNA expression. We developed new markers to aid transgene counter-selection in N. benthamiana , and demonstrate their efficacy for isolation of several genome-edited N. benthamiana lines. In Arabidopsis, we explore the limits of multiplexing by simultaneously targeting 12 genes by 24 sgRNAs. Perhaps surprisingly, the limiting factor in such higher order multiplexing applications is Cas9 availability, rather than recombination or silencing of repetitive sgRNA TU arrays. Through a combination of phenotypic screening and pooled amplicon sequencing, we identify transgene-free duodecuple mutant Arabidopsis plants directly in the T 2 generation. This demonstrates high efficiency of the zCas9i gene, and reveals new perspectives for multiplexing to target gene families and to generate higher order mutants.

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