*4.2. Cargo-Vectors for the CRISPR*/*Cas9 System*

Two kinds of vector systems are used in CRISPR/Cas9-mediated GE, such as a single-vector system and a binary-vector system. A binary-vector system has been utilized for many years because of its ability for fast primary testing. Any specific vector having several gRNAs and a Cas9 protein expression cassette already constructed in it can be applied for plant transformation. Different structural construct of gRNAs can be utilized for numerous Cas9 proteins to design a unique gRNA: Cas9 nuclease, which allows more accuracy and easiness in experimental design. A single vector harboring both expression cassettes of gRNA and Cas9 protein is becoming more promising. Generally, in a single-vector system, RNA polymerase III-driven promoters (U6/U3) are designed for gRNA expression, whereas ubiquitin and *CaMV35S* promoters based on RNA polymerase II are exploited for *Cas9* gene expression. Continuous advancements in CRISPR/Cas9 permit researchers to develop smarter vector systems to regulate expression of gRNA and the *Cas9* gene. Recently, some new adjustments were made in the single-vector system, such as single polymerase II and dual polymerase II promoters. Single polymerase II vectors are applied to govern the expression of gRNA and the *Cas9* gene at the same time, while the dual polymerase II vectors exploit two different promoters to drive the expression of gRNA and the *Cas9* gene. The addition of all these latest technologies in the CRISPR/Cas9 delivery system assists to decrease the vector length, which eventually, improves the transformation efficiencies [99].
