Apr 1, 2020

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

BioRxiv : the Preprint Server for Biology
K. BarthelJohannes Stuttmann

Abstract

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 SpCas9-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 ...Continue Reading

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