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 del

SUBMITTER: Kirchner TW 

PROVIDER: S-EPMC5609758 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature