RNA-guided CRISPR activation (CRISPRa) systems have been developed in plants. However, simultaneous activation of multiple genes remains challenging. Here, we develop a highly robust CRISPRa system working in rice, Arabidopsis and tomato, CRISPR-Act3.0, through systematically exploring different effector recruitment strategies and various transcription activators based on dSpCas9 (deactivated Streptococcus pyogenes Cas9). Based on CRISPR-Act3.0, we further develop a versatile CRISPR-Combo platform, based on a single Cas9 protein, for simultaneous genome editing (targeted mutagenesis or base editing) and gene activation in plants. We showcase CRISPR-Combo’s powerful applications for boosting plant genome editing. First, CRISPR-Combo is used to shorten the plant life cycle and reduce the efforts in screening transgene-free genome-edited plants by activation of a florigen gene in Arabidopsis. Next, we demonstrate accelerated regeneration and propagation of genome-edited plants by activation of morphogenic genes in poplar. Furthermore, we apply CRISPR-Combo to achieve rice regeneration without exogenous plant hormones, which is established as a novel method to enrich high-efficiency genome-edited plants. In conclusion, CRISPR-Combo is a versatile genome engineering tool with promising applications in crop breeding.
Keynote Address: Boosting plant genome engineering: from CRISPR-Act3.0 to CRISPR-Combo
Prof Yiping Qi, Associate Professor, University of Maryland, USA