Delivery of Cas9/sgRNA RNP into Wheat Microspores Using Synthetic CPP for Genome Editing and Gene Expression Modulation
Bilichak, A., et al., Delivery of Cas9/sgRNA RNP into Wheat Microspores Using Synthetic CPP for Genome Editing and Gene Expression Modulation, in CRISPR-Cas Methods, M.T. Islam, P.K. Bhowmik, and K.A. Molla, Editors. 2020, Springer US: New York, NY. p. 191-202.
Plain language summary
In this method chapter, we describe the protocol for non-transgenic transfection of wheat microspores with the dCas9-VP64 activator to induce the expression of embryogenesis-promoting genes.
Genome editing using sequence-specific nucleases has, undoubtedly, become one of the most important breakthroughs in biotechnology in recent years, allowing for rapid and efficient generation of crop varieties with novel or improved traits. Despite wide applicability of the technology, a challenge in bringing edited crops to market is the lack of regulatory alignment between jurisdictions (e.g., EU). Additionally, technical difficulties in efficient delivery of genome editing components into plant cells pose a problem for adoption of the technology by many groups, since transgenic pipelines and facilities need to be in place. Even with recent advancements in cell culture and plant regeneration, much work is still needed for reducing the burden of plant transformation. To address this, alternative methods for delivery of the genome editing machinery have been explored that potentially offer non-transgenic means to genome editing. In this chapter, we describe a novel method for delivery of in vitro purified Cas9/sgRNA complexes into wheat microspores for generation of homozygous edited plants. We provide guidelines for purification of Cas9 protein from bacterial culture, in vitro transcription of sgRNA, and delivery of the ribonuclear complex into the cells using cell-penetrating peptides. Additionally, we demonstrate utility of the technology through non-transgenic modulation of gene activity using the chimeric activators dCas9-VP64. With further optimization, the method has the potential to accelerate the development of cereals with novel traits by speeding up the editing process and by producing germplasm that potentially avoids regulation.