CRISPR–Cas9-mediated editing of starch branching enzymes results in altered starch structure in Brassica napus

Starch branching enzymes (SBEs) are one of the major classes of enzymes that catalyze starch biosynthesis in plants. Here, we utilized the clustered regularly interspaced short palindromic repeats–CRISPR associated protein 9 (CRISPR–Cas9)-mediated gene editing system to investigate the effects of SBE mutation on starch structure and turnover in the oilseed crop Brassica napus. Multiple single-guide RNA (sgRNA) expression cassettes were assembled into a binary vector and two rounds of transformation were employed to edit all six BnaSBE genes. All mutations were heterozygous monoallelic or biallelic, and no chimeric mutations were detected from a total of 216 editing events. Previously unannotated gene duplication events associated with two BnaSBE genes were characterized through analysis of DNA sequencing chromatograms, reflecting the complexity of genetic information in B. napus. Five Cas9-free homozygous mutant lines carrying two to six mutations of BnaSBE were obtained, allowing us to compare the effect of editing different BnaSBE isoforms. We also found that in the sextuple sbe mutant, although indels were introduced at the genomic DNA level, an alternate transcript of one BnaSBE2.1 gene bypassed the indel-induced frame shift and was translated to a modified full-length protein. Subsequent analyses showed that the sextuple mutant possesses much lower SBE enzyme activity and starch branching frequency, higher starch-bound phosphate content, and altered pattern of amylopectin chain length distribution relative to wild-type (WT) plants. In the sextuple mutant, irregular starch granules and a slower rate of starch degradation during darkness were observed in rosette leaves. At the pod-filling stage, the sextuple mutant was distinguishable from WT plants by its thick main stem. This work demonstrates the applicability of the CRISPR–Cas9 system for the study of multi-gene families and for investigation of gene-dosage effects in the oil crop B. napus. It also highlights the need for rigorous analysis of CRISPR–Cas9-mutated plants, particularly with higher levels of ploidy, to ensure detection of gene duplications.