Efficient generation of mutations mediated by CRISPR/Cas9 in the hairy root transformation system of Brassica carinata

Abstract

A protocol for the induction of site-directed deletions and insertions in the genome of Brassica carinata with CRISPR is described. The construct containing the Cas9 nuclease and the guide RNA (gRNA) was delivered by the hairy root transformation technique, and a successful transformation was monitored by GFP fluorescence. PAGE analysis of an amplified region, presumably containing the deletions and insertions, demonstrated up to seven different indels in one transgenic root and in all analyzed roots a wildtype allele of the modified gene was not detectable. Interestingly, many of these mutations consisted of relatively large indels with up to 112 bp. The exact size of the deletions was determined to allow an estimation whether the targeted gene was not functional due to a considerable deletion or a frame shift within the open reading frame. This allowed a direct phenotypic assessment of the previously characterized roots and, in fact, deletions in FASCICLIN-LIKE ARABINOGALACTAN PROTEIN 1 (BcFLA1)–a gene with an expression pattern consistent with a role in root hair architecture–resulted in shorter root hairs compared to control roots ectopically expressing an allele of the gene that cannot be targeted by the gRNA in parallel to the CRISPR construct. As an additional line of evidence, we monitored BcFLA1 expression with qPCR and detected a significant reduction of the transcript in roots with an active CRISPR construct compared to the control, although residual amounts of the transcript were detected, possibly due to inefficient nonsense-mediated mRNA decay. Additionally, the presence of deletions and insertions were verified by Sanger sequencing of the respective amplicons. In summary we demonstrate the successful application of CRISPR/Cas9 in hairy roots of B. carinata, the proof of its effectiveness and its effect on the root hair phenotype. This study paves the way for experimental strategies involving the phenotypic assessment of gene lesions by CRISPR which do not require germline transmission.

Hairy Root Cultures—A Versatile Tool With Multiple Applications

“…Aspergillus niger, cellulase (Srivastava et al, 2016) gene involved in HRs architecture (BcFLA1) in Brassica carinata (Kirchner et al, 2017). The possibility to generate HR clones harboring knock-out gene(s) will facilitate metabolic pathway understanding or modification.…”

Molecular Background of Pi Deficiency-Induced Root Hair Growth in Brassica carinata – A Fasciclin-Like Arabinogalactan Protein Is Involved

“…Plant cultivation and screening for transgenic roots was done as described in Kirchner et al (2017) for the CRISPR experiments. As many 2-cm root tips as possible were harvested from each transgenic root for expression analysis.…”

CRISPR-Cas9-mediated editing ofmyb28impairs glucoraphanin accumulation ofBrassica oleraceain the field

“…This is further supported by the highly targeted transcriptional changes of sulphur metabolism genes in the transcriptomes of these mutant plants. Studies have used CRISPR-Cas9 mediated genome editing to target multiple gene copies in the allotetraploid B. napus and B. carinata genome (Braatz et al, 2017;Okuzaki et al, 2018;Sun et al, 2018;Yang et al, 2017;Yang et al, 2018) (Kirchner et al, 2017) with few reported off-target effects. To date, the only use of RNA-mediated CRISPR-Cas9 genome editing in Brassica oleracea is that of the technique used here developed at the John Innes Centre and described in (Lawrenson et al, 2019;Lawrenson et al, 2015).…”