Nissle 1917 is an E. coli strain with probiotic properties, but for certain applications, it is necessary to knock out specific genes^[1]. Since CRISPR-Cas systems have high target DNA specificity and programmability, they can serve as gene editing tools^[2]. 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, unwanted escapers that do not undergo the intended editing frequently arise during CRISPR-Cas-mediated microbial genome editing, reducing editing efficiency^[3].

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 codon optimization, lowering escape rates to improve editing efficiency, and providing a reference for other genome editing tools. Herein, we constructed two Cas9 systems to test the gene editing efficiency in E. coli Nissle 1917, one of which was designed by Prof. LI QI^[3], and the other was redesigned by codon optimization.

 

Design 1:

The original Cas9 encoding plasmid has a lower copy of pSC101 origin. We selected the higher copy p15A origin to increase Cas9 expression. This allows the retention of functional Cas9 during host editing to ensure normal CRISPR-Cas system operation, reducing escape rates and improving editing efficiency. In addition, the incorporation of the λ-Red recombination system can enhance editing efficiency, so we constructed the p15A-Cas9-λ-Red plasmid (Figure 1).

 

 

Figure 1 Engineering frame of the p15A-Cas9-λ-Red plasmid.

 

Build 1:

To construct p15A-Cas9-λ-Red, we amplified p15A and Cas9-λ-Red using primers with homology arms and recombined them using a cloning kit to generate p15A-Cas9-λ-Red. After transformation, single colonies grew on LB plates. Colony PCR and sequencing verified the correct construction of the p15A-Cas9-λ-Red plasmid (Figure 2).

 

Figure 2 The construction results of p15A-Cas9-λ-Red.

(A) Colony PCR results. (B) Sequencing results.

 

Test 1:

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 3).

 

Figure 3 The SDS-PAGE result of p15A-Cas9-λ-Red protein expression.

 

Subsequently, we tested the induction of Cas9 expression 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 4).

 

Figure 4 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 5, p15A-Cas9-λ-Red achieved 100% knockout of gntT and 80% for lacZ in E. coli Nissle 1917.

 

Figure 5 Editing efficiency results of Cas9 in E. coli Nissle 1917.

 

Learn 1:

The use of a higher copy origin in E. coli Nissle 1917 effectively increased the Cas9 copy number and increased the editing efficiency to 100% for the gntT gene. However, the editing efficiency of the lacZ gene was only 80%, suggesting that there is room for improvement.

 

 

Design 2:

The Cas9 gene used in this project is from Streptococcus. Due to species differences, some rare E. coli tRNAs may be heavily used in the original sequence, causing slow host growth. Codon optimization avoids rare tRNA usage so bacteria can grow faster while expressing the same product. We named the codon-optimized Cas9 as Cas9-op and constructed the p15A-Cas9-op-λ-Red plasmid (Figure 6).

 

Figure 6 Expression frame of the p15A-Cas9-op-λ-Red plasmid.

 

Build 2:

To construct p15A-Cas9-op-λ-Red, we amplified p15A and Cas9-op-λ-Red fragments using primers with homology arms and recombined them using a cloning kit. Results showed successful amplification and construction verified by colony PCR and sequencing (Figure 7).

 

Figure 7 The construction results of p15A-Cas9-op-λ-Red.

(A) Transformation results. (B) Sequencing results.

 

Test 2:

Comparing to p15A-Cas9-λ-Red (BBa_K4876011), after Cas9 codon optimization, p15A-Cas9-op-λ-Red (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 further studied 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.

 

Figure 9 Editing efficiency results of Cas9-op with different lengths of homology arms in E. coli Nissle 1917.

 

Learn 2:

Using a higher copy origin and codon optimizing Cas9 effectively improved the editing efficiency of this CRISPR-Cas9 system on the gntT and lacZ genes in E. coli Nissle 1917 to 100%. 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. In the future, we will conduct more experiments to evaluate our Cas9-op tool on gene editing in order to expand its application for broader contribution.

 

[1] Buddenborg C., Daudel D., Liebrecht S., et al. Development of a tripartite vector system for live oral immunization using a gram-negative probiotic carrier [J]. Int J Med Microbiol, 2008, 298(1-2): 105-114.

[2] 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.

[3] 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.