CRISPR-Cas9 for Genome Editing and Genetic Engineering
CRISPR-Cas9 is a versatile tool for genetic engineering. This simple tool can be used for genomic sequence editing, transcriptional regulation, and other applications.
Design Vectors to Express gRNA-Cas9 in Plants
To express gRNA-Cas9 in plants, a plasmid vector (RGE vector) containing necessary components to express Cas9 and gRNA should be introduced into the plant cell. An example of plasmid construction for editing rice genes is available in our previous publication: (Xie and Yang, 2013).
Components of a RGE vector:
- RNA Polymerase III (Pol III) promoter and terminator to control transcription of gRNA
- gRNA with spacer sequence paired to targeting PAM site
- RNA Polymerase II (Pol II) promoter and terminator to control transcription of Cas9
- Cas9 coding sequence with nuclear localization signal (NLS)
Examples of promoters used in RGE vector construction:
- Pol II promoters: CaMV 35S, maize UBIQUITIN, rice ACTIN
- Pol III promoters: snoRNA U3, snoRNA U6