Besides genome editing, the CRISPR-Cas9-based platform provides a new way of engineering artificial transcription factors (ATFs). Multiplex of guide RNA (gRNA) expression cassettes holds a great promise for many useful applications of CRISPR-Cas9. In this chapter, we provide a detailed protocol for building advanced multiplexed CRISPR-dCas9-Activator/repressor T-DNA vectors for carrying out transcriptional activation or repression experiments in plants. We specifically describe the assembly of multiplex T-DNA vectors that can express multiple gRNAs to activate a silenced gene, or to repress two independent miRNA genes simultaneously in Arabidopsis. We then describe a "higher-order" vector assembly method for increased multiplexing capacity. This higher-order assembly method in principle allows swift stacking of gRNAs cassettes that are only limited by the loading capacity of a cloning or expression vector.
Keywords: Artificial transcription factor; CRISPR-Cas9; Gateway Cloning; Golden Gate assembly; Multiplex; Plant transcriptional regulation; Transcriptional activator; Transcriptional repressor.