Efficient CRISPR/Cas‐SF01 genome editing tools with high editing efficiency in allotetraploid oilseed rape

Mar 9, 2026Journal of integrative plant biology

Highly efficient genome editing tools for oilseed rape with multiple chromosome sets

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Abstract

Cas-SF01 demonstrates efficient genome editing in oilseed rape with an expanded PAM range.

Cas-SF01 is capable of cleaving at 5'-TTN-3' PAM sites, allowing for broader editing possibilities than conventional methods.
Editing outcomes from Cas-SF01 show considerable similarities to those achieved by the CRISPR-Cas9 system.
The system enables high-efficiency mutagenesis at multiple target sites, resulting in distinct phenotypes such as multilocular silique and male sterility.
Cas-SF01-derived cytosine base editors successfully produce targeted C-to-T base edits.
No off-target mutations were detected in the edited plants, indicating specificity in the editing process.

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CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 has been widely utilized for plant genome editing, but the protospacer adjacent motif (PAM) requirement limits its editing scope. CRISPR/Cas12i3 belongs to the type-VI Cas system that has gained extensive attention due to its smaller size and less restricted canonical TTN PAM sequence. In this study, we explored the newly developed Cas-SF01 system (Cas12i3 variant) for genome editing in oilseed rape. We established an efficient protoplast transformation system in oilseed rape to compare editing efficiency between Cas-SF01 and Cas9. Cas-SF01 shows cleavage activities at the tested 5'-TTN-3' PAM sites with editing outcomes sharing considerable similarities with the CRISPR-Cas9 system in protoplast. Cas-SF01 also induces high efficiency mutagenesis for multiple target sites in stable transformed oilseed rape lines, generating mutants with multilocular silique and male sterile phenotypes. Furthermore, Cas-SF01-derived cytosine base editors (CBEs) were developed to produce targeted C-to-T base edits. Compared to SpCas9, Cas-SF01 has an expanded PAM range and effectively recognizes TTN PAMs, which has substantially broadened the scope of editable sites within the rapeseed genome. No mutations were identified at the putative off-target sites among the edited plants. This study developed a robust, first-of-its-kind Cas12 system in the allotetraploid Brassica napus, expanding the scope of editing and enriching genome-editing toolkits for biological research and genetic improvement.