A Portal of CRISPR-Cas9 Mediated Genome Editing

CRISPR-Cas9 for genome editing and genetic engineering

CRISPR-Cas9 is a versatile tool for genetic engineering. This simple tool could be used for genomic sequence editing, transcriptional regulation and other potential 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 to edit rice genes could be found in our previous publication (Xie and Yang, 2013).

RGE vector backbone
    Components of a RGE vector:
  1. RNA Polymerase III (Pol III) promoter and Pol III terminator (Pol III Ter) to control the transcription of gRNA;
  2. gRNA with spacer sequence paired to targeting PAM-site;
  3. RNA polymerase II (pol II) promoter and Pol II terminator (Pol II Ter) to control the transcription of Cas9 gene;
  4. Cas9 with nuclear localization signal (NLS).
    Examples of promoters used to construct RGE vector:
  1. Pol II promoter: CaMV 35S promoter, maize UBIQUITIN promoter, rice ACTIN promoter, etc.
  2. Pol III promoter: snoRNA U3 promoter, snoRNA U6 promoter, etc.

CRISPR-PLANT is supported by Penn State and AGI.