Generation of Genomic Deletions (of Rig-I GENE) in Goat Primary Cell Culture Using CRISPR/CAS9 Method

ABSTRACT

CRISPR/Cas9 system is a natural immune system in prokaryotes protecting them from infectious viral or plasmid DNA invading the cells. This RNA-guided system can act as powerful tool for introducing genomic alterations in eukaryotic cells with high efficiency. In the present study, Rig-Igene is taken as model gene to study the efficiency of CRISPR/Cas9 system induced gene deletion in primary fibroblast cell culture. Rig-I(retinoic acid-inducible gene-1) is involved in regulating immune response in mammals. In this study, we optimized the CRISPR/Cas9 method for knocking out Rig-Igene in Goat primary fibroblasts by using a NHEJ pathway. Cells were screened for inactivation of the Rig-Igene and two positive clones were found out of thirty colonies screened. Thus, cells containing Rig-Igene inactivation could be achieved by CRISPR/Cas9 in goat fibroblast cells.

KEYWORDS:

• Immunity
• Genome editing
• CRISPR/cas9
• RIG-1

Acknowledgments

Shivani Malpotra and Ashutosh Vats contributed equally to this work. We are also grateful to Dr. Feng Zhang (MIT) for generously providing (px459) V2.0 plasmid.

Supplementary

Figure 1: Transfection efficiency of Goat fibroblasts: (A) Transfection of fibroblast with GFP expression plasmid (pacgfpi-NI) for transfection duration of 6 hrs and 12 hrs. (B) Graphical representation (imagej, NIH) of transfection efficiency based on duration showing higher efficiency with 6 hrs duration than 12 hrs.

Figure 2: Transfection efficiency depending upon concentration of Lipofectamine 2000, Showing constant fluorescence from 3 μg to 5 μg concentration.

Additional information

Funding

This work was supported by the National Dairy Research Institute, Karnal.