Part:BBa_K5382150:Design

considerations in the design process

In this study, we introduced a novel method for the efficient biosynthesis of Cas9 ribonucleoproteins (RNPs) using a refined E. coli expression system, specifically with the wild-type Escherichia coli</i> Nissle 1917 (EcN) strain. Based on our previous work [1], we have engineered an in vivo self-assembling plasmid designed for Cas9 RNP expression, where in the synthesis of both the Cas9 protein and guide RNA (gRNA) is governed by the Tac promoter, which is recognized by E. coli</i> RNA polymerase. Following transformation into the wild-type EcN, the plasmid facilitated the expression of Cas9 RNP. The purified Cas9 RNP was then analyzed through in vitro assays (Figure 1) to assess its capacity to cleave target DNA sequences.

Figure 1. Purification efficiency and in vitro activity verification experiment of Cas9 RNP.
Lane M: Pre-stained protein marker Lane;1: Target plasmid PCDNA3.1-Flag-PRDX4 Lane;2: pCold-Cas9-P4 experimental group;Lane3: SpeI single enzyme digestion;Lanes 4-7: Gradient elution with different concentrations of imidazole: 20 mM, 50 mM, 300 mM, 300 mM.
Lane M: DNA Marker;Lane 1: The target plasmid;Lane 2: cleaved plasmid by Cas9 RNP;Lane 3: Cleaved plasmid by Spe I.

Our experimental findings revealed that the purity of Cas9 ribonucleoproteins (RNPs), as determined by nickel column chromatography, was notably high and displayed adequate cleavage activity on target plasmids. However, the yield was suboptimal. Specifically, the use of the Tac promoter for Cas9 RNP synthesis yielded approximately 1 mg per liter of culture medium, which was markedly lower than anticipated. Our analysis indicates that the low yield may be due to the relatively weak activity of the Tac promoter, which likely resulted in reduced transcription of gRNA and, consequently, diminished assembly and enzymatic activity of the Cas9 RNP complexes. We noted that the WT EcN strain does not possess T7 RNA polymerase, which is necessary for recognizing the stronger T7 promoter. Consequently, to augment gRNA expression and enhance the assembly efficiency of Cas9 RNP complexes, we designed an experiment to incorporate the T7 RNA polymerase gene into the EcN genome. The re-designed expression plasmid was shown in Figure 2.

Figure 2. Expression plasmid of Cas9 RNP after promoter insertion.

We purified the engineered EcN strain and successfully isolated Cas9 RNPs utilizing the T7 promoter. Subsequently, the enzymatic cleavage activity of the isolated Cas9 RNPs was confirmed via in vitro assays (Figure 3), indicating an extraordinary nuclease activity. A comparative analysis of the yield of Cas9 RNPs produced using the T7 promoter versus the Tac promoter was performed and is illustrated in Figure 4. The results revealed that the expression level of Cas9 RNPs reached 8 mg per liter of culture medium, which significantly surpassed that achieved using the Tac promoter.

Figure 3. In vitro cleavage of the target plasmid.
Lane M DNA Marker ; Lane 1 Target plasmid ; Lane 2 Cleaved plasmid by XbaI; Lane 3 Cleaved plasmid by Cas9 RNP.

Figure 4. Comparative analysis of Cas9 RNP production driven by T7 and Tac promoters, subsequent to purification

Subsequently, we utilized a well-established engineering technique to produce outer membrane vesicles (OMVs) from genetically modified EcN, employing the lipid extruder method. The OMVs were subsequently purified and their size was characterized using transmission electron microscopy (TEM) and dynamic light scattering (DLS). Our findings confirm the successful assembly of Cas9 ribonucleoprotein (RNP)-loaded OMVs by the engineered EcN, with an average particle diameter of approximately 300 nm (Figure 5).

Figure 5. TEM image analysis and DLS analysis results of OMVs

Having confirmed the expression levels and enzymatic activity of Cas9 RNPs, was well as the OMS delivery system for these RNPs, we then investigate their potential applications in cellular genome editing (as detailed in the Experience section).