Nissle 1917 is an E. coli strain with probiotic properties, but for certain applications, it is necessary to knock out specific genes. Since CRISPR-Cas systems have high target DNA specificity and programmability, they can serve as gene editing tools. However, unwanted escapers that do not undergo the intended editing frequently arise during CRISPR-Cas-mediated microbial genome editing, reducing editing efficiency.
Therefore, this project aims to construct a highly efficient CRISPR-Cas9-based genome editing tool for E. coli Nissle 1917 by increasing Cas9 copy number and conducting Cas9 codon optimization, lowering escape rates to improve editing efficiency, and providing a reference for other genome editing tools.
Our experimental results mainly include:
(1) Construction of Cas9 plasmids, gRNA plasmids, and homology arms.
Ø Cas9: p15A-Cas9-λ-Red (p15A-Cas9), p15A-Cas9-op-λ-Red (p15A-Cas9-op), pColE1-Cas9-op-λ-Red (pColE1-Cas9-op)
Ø gRNA: gRNA-gntT, gRNA-lacZ
Ø Homology arms: gntT-HR, lacZ-HR
(2) Inducing protein expression of Cas9.
(3) Editing efficiency tests.
We compared our re-designed Cas9-op tool with the “old” Cas9 tool[1] which was designed by Prof. Li in order to evaluate the gene editing efficiency on E. coli Nissle 1917.
Figure 1 Schematic flow diagram of this experiment.
1. Construction of Cas9 Plasmids, gRNA Plasmids, and Homology Arms.
To construct a highly efficient CRISPR-Cas9-based genome editing tool, we selected the origins of replication p15A and ColE1 to increase the Cas9 copy number. The cas9 gene used in this study is from Streptococcus, causing slow growth of the E. coli host. Thus, we obtained Cas9-op by codon optimization to increase bacterial growth speed. We also incorporated the λ-Red recombination system to improve editing efficiency. After obtaining this optimized genome editing tool, we chose two genes, gntT, and lacZ, to test whether the editing efficiency was improved.
1) Construction of p15A-Cas9 plasmid.
To construct p15A-Cas9, we amplified p15A and Cas9 using primers with homology arms and recombined them using a cloning kit to generate p15A-Cas9. After transformation, single colonies grew on LB plates. Colony PCR and sequencing verified the correct construction of the p15A-Cas9 plasmid (Figure 2).
Figure 2 The construction results of p15A-Cas9.
(A) Colony PCR results. (B) Sequencing results.
2) Construction of p15A-Cas9-op plasmid.
To construct p15A-Cas9-op, we first codon-optimized the Cas9 gene (i.e. Cas9-op) to make it more suitable for E. coli host. Then, we amplified p15A, Cas9-op fragments using primers with homology arms and recombined them using a cloning kit to construct p15A-Cas9-op. After transformation, single colonies grew on LB plates. Colony PCR and sequencing result validated the correct construction of the p15A-Cas9-op plasmid (Figure 3).
Figure 3 The construction results of p15A-Cas9-op.
(A) Transformation results. (B) Sequencing results.
3) Construction of pColE1-Cas9-op plasmid--- Fail.
To construct pColE1-Cas9-op, we amplified ColE1 and Cas9-op using primers with homology arms and attempted recombination. Although fragments were amplified successfully, sequencing result showed the plasmid was not correctly assembled (Figure 4), likely due to homologous recombination errors or gene mutation. This issue is still pending deeper investigation and external professional opinions.
Figure 4 The construction results of pColE1-Cas9-op.
(A) Fragment amplification results. (B) Failed sequencing results.
4) Construction of gRNA-gntT
To construct gRNA-gntT, we linearized gRNA by digesting with BsaI. The annealed oligos with BsaI sites and gntT sequence were then ligated into linearized gRNA using T4 DNA ligase. After transformation, single colonies grew on LB plates. Colony PCR and sequencing verified the correct construction of the gRNA-gntT plasmid (Figure 5).
Figure 5 The construction results of the g gRNA-gntT plasmid.
(A) Transformation results. (B) Colony PCR results. (C) Sequencing results.
5) Construction of gRNA-lacZ
To construct gRNA-lacZ, we linearized gRNA by digesting with BsaI. The annealed oligos with BsaI sites and lacZ sequence were then ligated into linearized gRNA using T4 DNA ligase. After transformation, single colonies grew on LB plates. Colony PCR and sequencing verified the correct construction of the gRNA-lacZ plasmid (Figure 6).
Figure 6 The construction results of the g gRNA-lacZ plasmid.
(A) Transformation results. (B) Colony PCR results. (C) Sequencing results.
6) Amplification of Repair Homology Arms.
To obtain homology arms of gntT-HR and lacZ-HR, we amplified fragments of different lengths (100-500 bp) using gRNA-gntT and gRNA-lacZ as templates. The results are shown in Figure 7, we successfully amplified 100-500 bp repair homology arms.
Figure 7 Amplification results of homology arms.
(A) Amplification results of the 500 bp homology arms. (B) Amplification results of the 100-400 bp homology arms.
2. Inducing Protein Expression of Cas9
1) SDS-PAGE of Protein
To verify Cas9 protein, inducing expression and purification was conducted. The results showed that we successfully induced Cas9 protein expression (Figure 8).
Figure 8 The SDS-PAGE result of p15A-Cas9.
2) BSA Curves of Protein Expression by L-arabinose Induction
Subsequently, we tested the inductive effects of different L-arabinose concentrations on Cas9 expression. We quantified Cas9 concentrations based on a BSA standard curve (Figure 9A). The results showed that Cas9 expression increased with higher L-arabinose concentrations, indicating that we can control Cas9 levels by modulating L-arabinose addition (Figure 9B). By adjusting L-arabinose concentration levels, we can achieve controlled Cas9 production to balance editing efficiency and toxicity.
Figure 9 Results of Cas9 protein induction by L-arabinose.
(A) Standard curve of BSA concentration. (B) The curve of Cas9 concentration.
3. Editing Efficiency Test
1) Editing Efficiency Comparison: p15A-Cas9 and p15A-Cas9-op
We electroporated the Cas9 plasmids, gRNA plasmids, and homology arms (500 bp) into E. coli Nissle 1917. After 16 h growth, colony PCR identified transformants. Results showed p15A-Cas9 achieved 100% knockout of gntT and 80% for lacZ in E. coli Nissle 1917. After Cas9 codon optimization, p15A-Cas9-op knocked out both gntT and lacZ at 100% efficiency (Figure 10). Therefore, we could draw the conclusion that the editing efficiency of our optimized Cas9 tool is higher than that of the “old” Cas9 tool[1] which was designed by Prof. Li on E. coli Nissle 1917.
Figure 10 Editing efficiency results of p15A-Cas9 and p15A-Cas9-op in E. coli Nissle 1917.
2) Effect of the Length of Repair Homology Arms on Editing Efficiency: p15A-Cas9-op
We further verified the editing efficiencies of p15A-Cas9-op for the gntT gene and lacZ gene by adjusting the length of the repair homology arms. The results are shown in Figure 11, for the gntT gene, the editing efficiencies of the repair homology arm of 100-200 bp were 0, while it reached 100% when the lengths were extended to 300-400 bp. Similarly, for the lacZ gene, the editing efficiency of the repair homology arm of 100 bp was 0, which increased to 80% for 200-300 bp, and reached 100% for 400 bp. These results indicated that the length of the repair homology arm is crucial to the editing efficiency. To ensure high editing efficiency, the length of the repair homology arm should preferably be more than 300 bp.
Figure 11 Editing efficiency results of p15A-Cas9-op with different lengths of homology arms in E. coli Nissle 1917.
Summary
In summary, we successfully constructed optimized Cas9 plasmids (p15A-Cas9-op) and the gene editing efficiency on E. coli Nissle 1917 was evaluated by comparing to the “old” Cas9 plasmid (p15A-Cas9). Our results demonstrate utilizing higher copy origins of replication and codon optimizing Cas9 can effectively improve the editing efficiency of this CRISPR-Cas system on the gntT and lacZ genes in E. coli Nissle 1917. But, we also failed in construction of the plasmid, pColE1-Cas9-op. For this regard, we will further consult with more experts to seek their professional advice to modify our design in order to achieve higher editing efficiency.
[1] Li Q., Sun M., Lv L., et al. Improving the editing efficiency of crispr-cas9 by reducing the generation of escapers based on the surviving mechanism [J]. ACS Synthetic Biology, 2023, 12(3): 672-680.