Developing highly efficient CRISPR-Cas-based genome editing tools has been a research focus in recent years across various fields[1]. The development and optimization of such tools can facilitate the construction of engineered cell lines for diverse production needs while accelerating the exploration of biological systems. However, the generation of escapers is one of the critical limiting factors in developing efficient editing tools[2]. This project aims to construct a highly efficient CRISPR-Cas9-based genome editing tool by utilizing strategies of increasing Cas9 copy number and codon optimization. We selected the origins of replication p15A and ColE1 to increase Cas9 copy number, optimized the cas9 gene from Streptococcus, and incorporated the λ-Red recombination system to improve editing efficiency on the gntT and lacZ genes in E. coli Nissle 1917, providing a reference for other genome editing tools. Therefore, we contributed the following new parts:
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Part Contributions |
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Part Number |
Part Number |
Contribution Type |
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Cas9 |
New experimental data to an existing part |
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Lambda-Red |
New experimental data to an existing part |
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p15A-Cas9-λ-Red |
New part (by incorporating BBa_K2200005, BBa_K3333007 and p15A) |
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lacZ |
New experimental data to an existing part |
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gRNA-lacZ |
New part |
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gRNA-gntT |
New part |
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gntT |
New part |
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Cas9-op |
New part (by improving BBa_K2200005) |
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p15A-Cas9-op-λ-Red |
New part (by improving BBa_K4876011) |
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1. Add new experimental data to existing parts: BBa_K2200005 and BBa_K3333007.
The original Cas9 encoding plasmid (BBa_K2200005) has the pSC101 origin with a low copy number. We selected the higher copy p15A origin to increase Cas9 expression level. This allows the retention of functional Cas9 during host editing to ensure normal CRISPR-Cas system operation, reducing escape rates and improving editing efficiency. Furthermore, incorporating the λ-Red (BBa_K3333007) recombination system can enhance editing efficiency. Thus, we constructed the p15A-Cas9-λ-Red (BBa_K4876011) plasmid (Figure 1).
Figure 1 The construction results of p15A-Cas9-λ-Red.
(A) Colony PCR results. (B) Sequencing results.
To verify Cas9 expression in E. coli Nissle 1917, we transformed the constructed p15A-Cas9-λ-Red plasmid. After inducing expression, we lysed the cells by sonication and purified Cas9. The results showed that we successfully induced Cas9 protein expression (Figure 2).
Figure 2 The SDS-PAGE result of p15A-Cas9-λ-Red protein expression.
Subsequently, we tested the Cas9 expression induced by different concentrations of L-arabinose. The results showed that with higher concentrations of L-arabinose, the expression of Cas9 was higher, suggesting that we could control the level of Cas9 by regulating the addition of L-arabinose (Figure 3).
Figure 3 Results of Cas9 protein induction by L-arabinose.
(A) Standard curve of BSA concentration. (B) The curve of Cas9 concentration.
We electroporated p15A-Cas9-λ-Red along with gRNA-gntT/lacZ and gntT/lacZ-HR homology arms into E. coli Nissle 1917. After 16 h growth, colony PCR identified transformants. As shown in Figure 4, p15A-Cas9-λ-Red achieved 100% knockout of gntT and 80% for lacZ in E. coli Nissle 1917.
Figure 4 Editing efficiency results of Cas9 in E. coli Nissle 1917.
2. Add new experimental data to an existing part BBa_K2550201 and Create a new part BBa_K4876008
To edit the lacZ gene (BBa_K2550201) in E. coli Nissle 1917, we constructed the gRNA-lacZ (BBa_K4876008) plasmid. The lacZ gene is a common editing site, so testing editing efficiency at this site could demonstrate the versatility of our CRISPR-Cas9 system. As shown in Figure 5, we successfully constructed both plasmids.
Figure 5 The construction results of the gRNA-lacZ plasmid.
(A) Transformation results. (B) Colony PCR results. (C) Sequencing results.
3. Create new parts: BBa_K4876009 and BBa_K4876007
To edit the gntT gene (BBa_K4876009) in E. coli Nissle 1917, we constructed the gRNA-gntT (BBa_K4876007) plasmid. The gntT gene has relatively low editing efficiency, so testing on this site can strongly demonstrate the efficacy of our CRISPR-Cas9 system. As shown in Figure 6, we successfully constructed both plasmids.
Figure 6 The construction results of the g pEcgRNA-gntT plasmid.
(A) Transformation results. (B) Colony PCR results. (C) Sequencing results.
4. Create new parts: BBa_K4876005 and BBa_K4876010
The Cas9 protein sequence used in this study is from Streptococcus. Due to differences in species, some rare tRNAs in the original sequence may be heavily used in E. coli, causing slow host growth. Codon optimization avoids rare tRNA usage as much as possible so that bacteria can grow faster while expressing the same product. We named the codon-optimized Cas9 as Cas9-op (BBa_K4876005) and constructed the p15A-Cas9-op-λ-Red plasmid (BBa_K4876010) (Figure 7).
Figure 7 The construction results of p15A-Cas9-op-λ-Red.
(A) Transformation results. (B) Sequencing results.
Comparing to p15A-Cas9 (BBa_K4876011) which achieved 100% knockout of gntT and 80% for lacZ in E. coli Nissle 1917. After Cas9 codon optimization, p15A-Cas9-op (BBa_K4876010) knocked out both gntT and lacZ at 100% efficiency which is higher than p15A-Cas9 (Figure 8).
Figure 8 Editing efficiency results of p15A-Cas9 and p15A-Cas9-op in E. coli Nissle 1917.
We also verified the editing efficiencies of p15A-Cas9-op-λ-Red for the gntT gene and lacZ gene by adjusting the length of the repair homology arms. The results are shown in Figure 9, for the gntT gene, the editing efficiencies of the repair homology arms of 100-200 bp were 0, while it reached 100% when the lengths were extended to 300-500 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 400-500 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 9 Editing efficiency results of Cas9-op with different lengths of homology arms in E. coli Nissle 1917.
[1] Vento J.M., Crook N., Beisel C.L. Barriers to genome editing with CRISPR in bacteria [J]. J Ind Microbiol Biotechnol, 2019, 46(9-10): 1327-1341.
[2] 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.