Application of Cas12a and nCas9-activation-induced cytidine deaminase for genome editing and as a non-sexual strategy to generate homozygous/multiplex edited plants in the allotetraploid genome of tobacco
Protoplasts can be used for genome editing using several different CRISPR systems, either separately or simultaneously, and that the resulting mutations can be recovered in regenerated non-chimaeric plants.
Protoplast transfection and regeneration systems are useful platforms for CRISPR/Cas mutagenesis and genome editing. In this study, we demonstrate the use of Cpf1 (Cas12a) and nCas9-activation-induced cytidine deaminase (nCas9-Target-AID) systems to mutagenize Nicotiana tabacum protoplasts and to regenerate plants harboring the resulting mutations. We analyzed 20 progeny plants of Cas12a-mediated phytoene desaturase (PDS) mutagenized regenerants, as well as regenerants from wild-type protoplasts, and confirmed that their genotypes were inherited in a Mendelian manner. We used a Cas9 nickase (nCas9)-cytidine deaminase to conduct C to T editing of the Ethylene receptor 1 (ETR1) gene in tobacco protoplasts and obtained edited regenerates. It is difficult to obtain homozygous edits of polyploid genomes when the editing efficiency is low. A second round of mutagenesis of partially edited regenerants (a two-step transfection protocol) allowed us to derive ETR1 fully edited regenerants without the need for sexual reproduction. We applied three different Cas systems (SaCas9, Cas12a, and nCas9-Traget AID) using either a one-step or a two-step transfection platform to obtain triply mutated and/or edited tobacco regenerants. Our results indicate that these three Cas systems can function simultaneously within a single cell.
KeywordsCRISPR/Cas Chimeric plants Cas12a Cytidine deaminase Polyploid genome editing
We acknowledge Drs. Masaki Endo and Seiichi Toki of the Plant Genome Engineering Research Unit, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, for Cas12a plasmids. This research was supported by Academia Sinica, Innovative Translational Agricultural Research Administrative Office (AS-KPQ-107-ITAR-10), and the Ministry of Science and Technology (105-2313-B-001 -007 -MY3), Taiwan.
All authors conceived and designed the experiments. CSL, CTH, YJC, QWC, YHY, and WFH performed targeted mutagenesis analysis. CTH and CSL conducted protoplast regeneration. CTH, YJC, QWC, YHY, QWC, FHW, and WFH performed molecular biology experiments. CSL, LYL, and SBG interpreted the data. CSL, LYL, and SBG wrote the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests.
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