Optimized Plasmid Construction Strategy for Cas9

Abstract

Background/Aims: The target genome editing technology not only plays an important role in basic biology studies but also holds a great promise for potential clinical applications. The new generation of engineered nuclease RGEN (RNA Guided EndoNuclease) is much easier to construct and modify, and attracts more attentions. In the current study, we compared different plasmid construction strategies of Cas9-gRNA (guide RNA). Methods: Different plasmid construction strategies of Cas9-gRNA were compared. And more modifications were introduced into the plasmid construction strategy. Results: The plasmid construction efficiency of expressing the gRNA and Cas9 in one plasmid was lower than expressing them in two separate plasmids. However, they showed the similar genome editing efficiency. We further introduced the Golden-gate assembly and blue-white screening approaches into the Cas9-gRNA construction procedures, without the process of vector digestion and gel purification. Conclusions: Combing with the optimized gRNA structure (gRNA-BL) we identified before, we established one more cost-effective, time-saving and efficient plasmid construction strategy for Cas9-gRNA.

Keywords

• RNA guided endonuclease
• Genome editing
• Cas9
• Guide RNA
• gRNA