Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA

Abstract

Editing plant genomes is technically challenging in hard-to-transform plants and usually involves transgenic intermediates, which causes regulatory concerns. Here we report two simple and efficient genome-editing methods in which plants are regenerated from callus cells transiently expressing CRISPR/Cas9 introduced as DNA or RNA. This transient expression-based genome-editing system is highly efficient and specific for producing transgene-free and homozygous wheat mutants in the T0 generation. We demonstrate our protocol to edit genes in hexaploid bread wheat and tetraploid durum wheat, and show that we are able to generate mutants with no detectable transgenes. Our methods may be applicable to other plant species, thus offering the potential to accelerate basic and applied plant genome-engineering research.

Genomic innovation for crop improvement

“…Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 technologies are being applied to crops for genome editing. The delivery of Cas9 and DNA templates to crop genomes can be carried out transiently 72 or using a viral vector 73 , without an intermediate transgenic step. This challenges the present definitions of genetic engineering according to European Union regulations.…”

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

“…The CRISPR-Cas system can be stably or transiently transfected into the plant cells as nucleic acids (plasmids or DNA fragments) [6,73] or pre-assembled ribonucleoprotein complexes (RNPs) [74][75][76].…”

“…Very often, to increase the proportion of Cas9 protein effectively addressed to the nucleus, one or two nuclear localization signals (such as A. tumefaciens bipartite VirD2 T-DNA border endonuclease carboxyl terminal NLS, or the monopartite Simian virus 40 NLS) are incorporated into the expression cassettes at the amino and/or carboxyl termini of the Cas9 open reading frame [12,73,75,88].…”

“…Using Cas9 RNA or protein presents the advantage of not having to work on optimizing expression by defining sequences codon-optimized for the host plant and selecting appropriate promoters. Preassembled complexes of purified Cas9 protein and guide RNA (RNPs) were transfected into protoplasts of Arabidopsis thaliana, Nicotiana attenuata, Petunia x hybrida, grapevine, apple, lettuce and rice [74,76,95] and bombarded into immature embryos of bread and durum wheat [73,96], and maize [75]. In all cases, RNPs showed good cleavage efficiency (4-46%), with (when tested) no detectable mosaicism, suggesting a very early action of the nuclease before the first cell division [74,76].…”

“…Transient expression of the CRISPRCas9 system was also obtained by bombarding immature embryos of bread and durum wheats with in vitro transcribed sgRNAs and Cas9 RNAs. Mutagenesis frequency was lower (1.1%) than with DNA-based transient or stable transformation performed in the same conditions (about 3%), probably indicative of the instability of the RNAs in the plant cells [73]. Nevertheless, the rapid degradation of the RNAs is considered beneficial as it reduces the time during when the genome is exposed to nuclease activity, which limits the risk of generating off-target mutations [74].…”