Part:BBa_K5382150:Design

Cas9 ribonucleoproteins(gRNA PRDX4)-Co-expression and self-assembly of RNPs

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 4514
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Relevant factors to consider in the design process

In this study, we introduced a creative method for the efficient biosynthesis of Cas9 ribonucleoproteins (RNPs) using a refined E. coli expression system, specifically with the wild-type Escherichia coli 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 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 enzyme activity assay (Figure 1) to assess its capacity to cleave target DNA sequences.

Figure 1. Purification efficiency and in vitro activity verification experiments of Cas9 RNP.

a. Lane M: Pre-stained protein marker; Lane 1: Target plasmid PCDNA3.1-Flag-PRDX4; Lane2: 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.
b. 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 reveal that the purity of Cas9 ribonucleoproteins (RNPs), as determined by nickel column chromatography, was approximately 80% 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. The WT EcN strain does not possess T7 RNA polymerase, which is necessary for recognizing the stronger T7 promoter. Consequently, to enhacne the gRNA expression and the assembly efficiency of Cas9 RNP complexes, we then designed an experiment to incorporate the T7 RNA polymerase gene into the EcN genome (Figure 2).

Figure 2. The schematics of the expression plasmid for Cas9 RNP with a T7 promoter

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 nuclease assay (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 demonstrated that the expression level of Cas9 RNPs reached 8 mg per liter of culture medium, representing an approximately 8-fold increase compared to 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 using T7 and Tac promoters after purification.

Next, we utilized a well-established engineering technique to generate 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 RNP-loaded OMVs by the engineered EcN, with an average particle diameter of approximately 100 nm (Figure 5).

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

After confirming the expression levels and enzymatic activity of Cas9 RNPs, as well as evaluating the efficiency of their delivery system (OMS), we proceeded to explore their potential applications in cellular genome editing, which are elaborated in the Experimental section.

Source

We have achieved the co-expression of Cas9 enzyme and its related guide RNA(gRNA) in Escherichia coli Nissle 1917. The Cas9 protein is derived from Streptococcus pyogenes (S. pyogenes Cas9, SpCas9). The gRNA and Cas9 protein expression sequences in the system are constructed on the same plasmid, and the plasmid backbone is the pCold vector. The gRNA is artificially designed according to the sequence of the targeted gene PRDX4 and can specifically bind to its sequence.

References

[1] Qiao J, Li W, Lin S, Sun W, Ma L, Liu Y. Co-expression of Cas9 and single-guided RNAs in Escherichia coli streamlines production of Cas9 ribonucleoproteins. Commun Biol. 2019; 2:161.Published 2019 May 3. doi:10.1038/s42003-019-0402-x