Difference between revisions of "Part:BBa K5382150:Design"

Revision as of 09:37, 30 September 2024

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).

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-===Detail considerations in the design process===
+===considerations in the design process===
-In the initial design, we successfully achieved the co-expression of Cas9 enzyme and its associated guide RNA in E. coli, but later we found that this method still has some limitations, mainly in the low yield and long purification time.
+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.<br>
-In order to improve the yield of Cas9 RNPs, we introduced the CL7 label on the n-terminal of the original Cas9. The CL7 tag can be easily recognized by human rhinovirus (HRV) 3C protease cutting at 16°C for 3 hours. In addition, to prevent contamination of the 3C protease in the final sample, we used an engineered CL7-labeled HRV 3C protease. In the expression of sgRNA, we use T7 promoter to obtain higher expression efficiency
+https://static.igem.wiki/teams/5382/part-pictures/11.png<br>'''Figure 1.'''     Purification efficiency and in vitro activity verification experiment of Cas9 RNP.<br>
-The pCold-CL7-Cas9 expression plasmid scheme is shown in the figure.
+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.<br>Lane M: DNA Marker;Lane 1: The target plasmid;Lane 2: cleaved plasmid by Cas9 RNP;Lane 3: Cleaved plasmid by Spe I.<br>
-https://static.igem.wiki/teams/5382/part-pictures/little-sp.png<br>'''Figure 1.'''    The pCold-CL7-Cas9 co-expression plasmid.<br>
-According to the experimental design, when IPTG was added, sgRNA molecules were transcribed in large quantities in Escherichia coli, while CL7-Cas9 fusion protein was also expressed simultaneously in Escherichia coli.
-The final results showed that the Cas9 RNPs yield was increased to ~40 mg/L using LB medium, which was 4 times higher than the existing method.
-In terms of purification methods, we introduced an ultra-high affinity CL7/ Im7 system, which helped us achieve one-step purification of Cas RNPs in half a day. Compared with Cas9 RNPs purified by Ni-NTA affinity column, the purity of Cas9 RNPs purified by Im7 column was improved from ~58% to ~89% based on gray scale scan analysis (Figure 2).
-https://static.igem.wiki/teams/5382/part-pictures/middle-sp-plus.png<br>'''Fig, 2 a.''' A total of 12% SDS-PAGE of Cas9 RNP (10 μg, red square) and Cas12a RNP (5ug. blue square) purified by Ni-NTA affinity column or by Im7 column. The original uncropped gels were shown in Supplementary Fig, 7. '''b''' The target Cas RNPs' purity was validated from three individual batches of
-purification by gray scanning analysis (ImageJ) of the SDS-PAGE. Data are shown as the mean ± SD<br>
-The various bands visible on the gel of Ni-NTA purified Cas RNPs are proteins from E. coli itself. The purity of target Cas RNPs can be improved by gradient elution of imidazole with different concentration and purification by gel filtration. Importantly, reproducible results were observed when Cas9 RNPs were prepared with different Sgrnas (supplementary Figure 7). In addition, there is no difference between batches in the production of the same Cas9 RNPs. Notably, by coupling the recombinant Im7 enzyme to agarose beads, we simply prepared Im7 affinity columns that can be repeatedly regenerated without losing the binding affinity to CL7 affinity tag16.
-With our method, no RNase inhibitors are required during the entire purification and storage process. The resulting Cas9RNP is very stable and can be stored at -20 °C for 9 months without activity changes. From this, we propose that sgRNAs transcribed in E. coli can somehow bind tightly to newborn Cas9, helping Cas9 fold into a stable conformation and protecting sgRNAs from nuclease-mediated degradation.
-===Supplementary experimental results===
+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.<br>
-. Purification and in vitro activity verification of Cas9 RNP in EcN
-https://static.igem.wiki/teams/5382/part-pictures/crsipr1.png<br>'''Figure 3.'''   The mutant structure compared to the Wild type Ceres.<br>
-'''a.''' Lane M: three-color prestain protein Marker
+https://static.igem.wiki/teams/5382/part-pictures/little-sp.png<br>'''Figure 2.''' Expression plasmid of Cas9 RNP after promoter insertion.
-Lane 1: break bacteria precipitate Lane 2: Bacteria-breaking supernatant Lane 3: Flow eluent after binding with nickel beads
-Lane 4-7: different concentrations of imidazole gradient eluents '''b.''' Lane M:DNA Marker Lane 1: Target plasmid pCDNA3.1-Flag-PRDX4 Lane 2: pCold-Cl7-Cas9-P4 experimental group Lane 3:spel single enzyme digestion
+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.
+https://static.igem.wiki/teams/5382/part-pictures/13.png<br>'''Figure 3.''' In vitro cleavage of the target plasmid.<br>
+Lane M DNA Marker ; Lane 1 Target plasmid ; Lane 2 Cleaved plasmid by XbaI; Lane 3 Cleaved plasmid by Cas9 RNP.
+https://static.igem.wiki/teams/5382/part-pictures/14.png<br>'''Figure 4.'''  Comparative analysis of Cas9 RNP production driven by T7 and Tac promoters, subsequent to purification<br>
+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).<br>
+https://static.igem.wiki/teams/5382/part-pictures/15.png<br>'''Figure 5.''' TEM image analysis and DLS analysis results of OMVs<br>
+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).