Expanding the targeting scope of CRISPR/Cas9-mediated genome editing by Cas9 variants in Brassica

CRISPR/Cas9, presently the most widely used genome editing technology, has provided great potential for functional studies and plant breeding. However, the strict requirement for a protospacer adjacent motif (PAM) has hindered the application of the CRISPR/Cas9 system because the number of targetable genomic sites is limited. Recently, the engineered variants Cas9-NG, SpG, and SpRY, which recognize non-canonical PAMs, have been successfully tested in plants (mainly in rice, a monocot). In this study, we evaluated the targeted mutagenesis capabilities of these Cas9 variants in two important Brassica vegetables, Chinese cabbage (Brassica rapa spp. pekinensis) and cabbage (Brassica oleracea var. capitata). Both Cas9-NG and SpG induced efficient mutagenesis at NGN PAMs, while SpG outperformed Cas9-NG at NGC and NGT PAMs. SpRY achieved efficient editing at almost all PAMs (NRN > NYN), albeit with some self-targeting activity at transfer (T)-DNA sequences. And SpRY-induced mutants were detected in cabbage plants in a PAM-less fashion. Moreover, an adenine base editor was developed using SpRY and TadA8e deaminase that induced A-to-G conversions within target sites using non-canonical PAMs. Together, the toolboxes developed here induced successful genome editing in Chinese cabbage and cabbage. Our work further expands the targeting scope of genome editing and paves the way for future basic research and genetic improvement in Brassica.