Boosting genome editing in perennial plants by CRISPR-Combo mediated morphogenic gene activation

A major bottleneck in genome editing of many perennial plants is their recalcitrance to transformation and regeneration. To boost genome editing in such perennial crops, transcriptional reprogramming of morphogenic genes is introduced by CRISPR-Combo, a versatile system for simultaneous genome editing and transcriptional activation in plant cells. In potato, we screen 17 morphogenic genes and identify 4 genes (WOX11/12, ARF5, ABI3-1, and ABI3-2) that promote regeneration of genome-edited hairy roots, and 3 of the 4 genes are also found to boost shoot regeneration by Agrobacterium-mediated stable transformation. Similarly, screening of 10 morphogenic genes in citrus leads to the identification of 5 genes (BBM3, FUS3, IPT1, SERK1, and STM) that enhance plant regeneration upon activation. In wild strawberry, we demonstrate that simultaneous activation of Baby Boom genes (BBM1 and BBM2) or of GRF3 and GIF1 reduces the generation time of genome-edited plants by over one month. Moreover, in poplar, we show that simultaneous activation of WUS and WOX11 synergistically promotes plant regeneration without exogenous plant hormones, which leads to a protocol of generating genome-edited poplar shoots in less than one month. Collectively, this study provides efficient strategies for boosting genome editing in four perennial crops.