AUTHOR=Jedličková Veronika , Mácová Kateřina , Štefková Marie , Butula Jan , Staveníková Jana , Sedláček Marek , Robert Hélène S. 

TITLE=Hairy root transformation system as a tool for CRISPR/Cas9-directed genome editing in oilseed rape (Brassica napus)

JOURNAL=Frontiers in Plant Science

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2022.919290

DOI=10.3389/fpls.2022.919290

ISSN=1664-462X

ABSTRACT=<p>Our study examined the mutation efficiency of the CRISPR/Cas9 method for tryptophan aminotransferase <italic>BnaTAA1</italic> genes involved in the auxin biosynthesis pathway. We made nine CRISPR/Cas9 constructs with various promoters driving the expression of a Cas9 from <italic>Staphylococcus aureus</italic> (SaCas9) or a plant-codon-optimized <italic>Streptococcus pyogenes</italic> Cas9 (pcoCas9). We developed a fast and efficient system for evaluating the variety and frequency of mutations caused by each construct using <italic>Brassica napus</italic> hairy roots. We showed that pcoCas9 is more efficient in mutating the targeted loci than SaCas9 and the presence of the NLS signal enhanced the chance of mutagenesis by 25%. The mutations were studied further in regenerated lines, and we determined the <italic>BnaTAA1</italic> gene expression and heritability of the gene modifications in transgenic plants. Hairy root transformation combined with CRISPR/Cas9-mediated gene editing represents a fast and straightforward system for studying target gene function in the important oilseed crop <italic>B. napus</italic>.</p>