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.

Rapid Identification of Genome-Edited Mesenchymal Stem Cell Colonies via Cas9

“…Thus, we have tried to introduce IL-10 to the human genome safe harbor AASV1 site [19]. The gRNA structure and Cas9 system have been demonstrated before (Figure 2A) [16,17]. The donor structure and target sequence were obtained from SBI (Figure 2A).…”

“…Human IL-10 was PCR amplified and cloned into the donor plasmid according to the manufacturer's instructions. The gRNA target sequence (5′-GGGGCCACTAGGGACAGGAT-3′) was cloned into our Cas9-gRNA system as described before [16,17]. Plasmids were purified by PureYield ™ Plasmid Maxiprep System (Promega).…”

“…We found that lysis buffer containing Tween 20 and proteinase K (PK) was the most efficient approach to release DNA from only ten cells, which was sensitive enough for PCR detection. Previously, we had identified one optimized guide RNA structure with higher genome-editing efficiency and established one more efficient plasmid construction strategy for Cas9-gRNA [16,17]. We then combined these techniques to insert the immune suppressive gene IL-10 in the AAVS1 site, the safe harbor locus of the human genome [18,19].…”