597 / 2019-04-19 13:56:32

Using two-step transfections and multiple CRISPR nucleases to generate homozygous multiplex gene editings in the allotetraploid genome of tobacco

CRISPR,Genome editing,Protoplast

From models to crops > From models to crops

Protoplast transfection and regeneration systems are useful platforms for CRISPR/Cas mutagenesis and genome editing. In this report, 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 mutagenized and wild-type regenerants 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. Some progeny of the resulting regenerants inherited the mutant genotypes. However, some edited or mutated alleles were not passed on to
progeny plants. A two-step transfection protocol allowed us to derive ETR1 edited regenerants without the need for sexual reproduction. We applied three different Cas systems (SaCas9, Cas12a, and nCas9-Traget AID) in either a one-step or a two-step transfection platform to obtained triply mutated and/or edited tobacco regenerants. Our results indicate that these three Cas systems can function simultaneously within a single cell.