CRISPR-Cas9 system is one sequence-specific nuclease (SSN) that has several advantages over zinc finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN), such as multiplex genome editing. With multiplex genome editing, CRISPR-Cas9 becomes a preferred SSN to edit many quantitative trait loci (QTL) simultaneously for trait improvement in major crops. A multiplexed CRISPR system is also important for deletion of a large fragment within a chromosome, analysis of the function of gene families, exon exchange, gene activation, and repression. Therefore, assembly of several single guide RNAs (sgRNAs) into one binary vector is the main step in multigene editing by CRISPR-Cas9. Different vector construction methods have been practiced including Golden Gate assembly. This chapter provides a detailed protocol for the construction of a T-DNA binary vector for expressing Cas9 and three sgRNAs for simultaneous targeting of three QTL genes for improving seed trait in rice.
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