Part:BBa_K5382150:Design

Detail 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 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 The pCold-CL7-Cas9 expression plasmid scheme is shown in the figure.
Figure 1. The pCold-CL7-Cas9 co-expression plasmid.
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 (supplementary Table 1, see supplementary information). 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).
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
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

1. Purification and in vitro activity verification of Cas9 RNP in EcN
Figure 3. The mutant structure compared to the Wild type Ceres.
a. Lane M: three-color prestain protein Marker 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