CRISPR–Act3.0 for highly efficient multiplexed gene activation in plants

RNA-guided CRISPR activation (CRISPRa) systems have been developed in plants. However, the simultaneous activation of multiple genes remains challenging. Here, we develop a highly robust CRISPRa system working in rice, Arabidopsisand tomato, CRISPR–Act3.0, through systematically exploring different effector recruitment strategies and various transcription activators based on deactivated Streptococcus pyogenesCas9 (dSpCas9). The CRISPR–Act3.0 system results in fourfold to sixfold higher activation than the state-of-the-art CRISPRa systems. We further develop a tRNA–gR2.0 (single guide RNA 2.0) expression system enabling CRISPR–Act3.0-based robust activation of up to seven genes for metabolic engineering in rice. In addition, CRISPR–Act3.0 allows the simultaneous modification of multiple traits in Arabidopsis, which are stably transmitted to the T3 generations. On the basis of CRISPR–Act3.0, we elucidate guide RNA targeting rules for effective transcriptional activation. To target T-rich protospacer adjacent motifs (PAMs), we transfer this activation strategy to CRISPR–dCas12b and further improve the dAaCas12b-based CRISPRa system. Moreover, we develop a potent near-PAM-less CRISPR–Act3.0 system on the basis of the SpRY dCas9 variant, which outperforms the dCas9–NG system in both activation potency and targeting scope. Altogether, our study has substantially improved the CRISPRa technology in plants and provided plant researchers a powerful toolbox for efficient gene activation in foundational and translational research.