Epsilon Archive for Student Projects

Abstract

The accumulation of antinutritional compounds such as phytic acid, glucosinolates and sinapine limits the nutritional and industrial value of rapeseed (Brassica napus). CRISPR/Cas9 ribonucleoprotein (RNP)-mediated editing has been reported as a precise and transgene-free strategy for targeted gene modification without impairing plant performance. This approach has been successfully used in editing in B. napus for reducing seed antinutrients in my supervisor’s lab where many edited lines have been generated with single target gene mutation in single line. In this Master project, I tried to stack the mutated genes into same single lines by crossing. The genes concerned included BnX.SGT, BnREF1, BnGTR1, BnITPK4 and BnGTR2. Amplicon sequencing of SGT and REF1 progeny confirmed stable inheritance of frameshift mutations in the F₁ generation, mostly in heterozygous form. These results demonstrate that CRISPR RNP-mediated edits can be reliably transmitted and stacked through conventional breeding. Moreover, I have also evaluated mutation lines generated by multiplex CRISPR editing (X5) and ITPK4 mutant lines through biochemical analysis, sequencing, and seed germination tests. SGT lines showed no measurable reduction in sinapine, and sequencing of the lowest-sinapine plants revealed no CRISPR-induced edits at the sgRNA target region, indicating natural variation rather than editing effects. Future studies should therefore perform comprehensive genotyping of all lines before biochemical evaluation. In contrast, ITPK4-edited line S2 exhibited up to 23.6 % lower phytic acid compared to wild type, demonstrating the potential of these lines for effective gene editing to reduce phytic acid accumulation. However, expanded line evaluation is needed to confirm stable reductions and assess the consistency of this effect across multiple genetic backgrounds. Importantly, germination assays confirmed that CRISPR editing did not compromise seed viability, supporting its safe integration into breeding programs. Overall, this study demonstrates the potential of CRISPR/Cas9 RNP-mediated editing to generate heritable allelic variation in polyploid crops, providing a framework for reducing seed antinutrients. However, multi-generational evaluation under field conditions will be essential to confirm whether these edits translate into stable reductions and consistent agronomic performance.