Format

Send to

Choose Destination
Plant Mol Biol. 2019 Aug 10. doi: 10.1007/s11103-019-00907-w. [Epub ahead of print]

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.

Author information

1
Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan.
2
Department of Biological Sciences, Purdue University, West Lafayette, IN, 47907-1392, USA.
3
Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan. cslin99@gate.sinica.edu.tw.

Abstract

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.

KEYWORDS:

CRISPR/Cas; Cas12a; Chimeric plants; Cytidine deaminase; Polyploid genome editing

PMID:
31401729
DOI:
10.1007/s11103-019-00907-w

Supplemental Content

Full text links

Icon for Springer
Loading ...
Support Center