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.

KEY MESSAGE: 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.

Characterization of Arabidopsis glutamine phosphoribosyl pyrophosphate amidotransferase-deficient mutants.

Using a transgene-based screening, we previously isolated several Arabidopsis mutants defective in protein import into chloroplasts. Positional cloning of one of the loci, CIA1, revealed that CIA1 encodes Gln phosphoribosyl pyrophosphate amidotransferase 2 (ATase2), one of the three ATase isozymes responsible for the first committed step of de novo purine biosynthesis. The cia1 mutant had normal green cotyledons but small and albino/pale-green mosaic leaves. Adding AMP, but not cytokinin or NADH, to plant liquid cultures partially complemented the mutant phenotypes. Both ATase1 and ATase2 were localized to chloroplasts. Overexpression of ATase1 fully complemented the ATase2-deficient phenotypes. A T-DNA insertion knockout mutant of the ATase1 gene was also obtained. The mutant was indistinguishable from the wild type. A double mutant of cia1/ATase1-knockout had the same phenotype as cia1, suggesting at least partial gene redundancy between ATase1 and ATase2. Characterizations of the cia1 mutant revealed that mutant leaves had slightly smaller cell size but only half the cell number of wild-type leaves. This phenotype confirms the role of de novo purine biosynthesis in cell division. Chloroplasts isolated from the cia1 mutant imported proteins at an efficiency less than 50% that of wild-type chloroplasts. Adding ATP and GTP to isolated mutant chloroplasts could not restore the import efficiency. We conclude that de novo purine biosynthesis is not only important for cell division, but also for chloroplast biogenesis.