Using NgAgo to edit the genes of Mycobacterium smegmatis

NgAgo-F is the PIWI domain of NgAgo, which is approximately 717 bp in length and mediates bacterial genome editing (Fig. 1A). We codon-optimized the sequence of the NgAgo-F gene to make NgAgo-F much easier to express and function in M. smegmatis. Using homologous recombination, we successfully inserted Flag-NgAgo-F driven by the Hsp60 promoter into the shuttle plasmid pKC1139, which contains a temperature-sensitive pSG5 replication system and a cassette encoding the aac(3)-Ivan gene for apramycin resistance that can be subsequently lost through temperature treatment (18). Through this method, the pKC1139-Hsp60-Flag-NgAgo-F (pKHNgAgo-F) plasmid was constructed (Fig. 1B). Then, upstream and downstream sequences, with superfolder green fluorescent protein (sfGFP) between them, were introduced into the pKHNgAgo-F plasmid. A schematic diagram of the constructed knockout plasmid and the knockout principle is shown in Fig. 1C.

The solubility of the NgAgo-F protein is critical to its function. To verify the solubility of NgAgo-F, we first constructed the pET28a-His-NgAgo-F plasmid via the homologous recombination method. The plasmid was subsequently transformed into BL21(DE3) cells. His-NgAgo-F, a 31.2 kDa fusion protein, was successfully expressed and purified (Fig. 2A), demonstrating that NgAgo-F is soluble in E. coli. To further confirm whether the NgAgo-F protein is soluble in M. smegmatis, we transferred pKHNgAgo-F into the wild-type (WT) strain and obtained the strain pKHNgAgo-F::WT. Western blotting was performed using an anti-Flag antibody. The NgAgo-F protein was detected in the supernatant, confirming that the NgAgo-F protein is also soluble in M. smegmatis (Fig. 2B). These results indicate that NgAgo-F can achieve genome editing in M. smegmatis.

To determine whether the NgAgo-F tool can achieve genome deletion in M. smegmatis, we first constructed a plasmid for glnR knockout. To ensure high cleavage efficiency, we recombined 800 bp upstream and 800 bp downstream homology arms into the pKHNgAgo-F plasmid and obtained the plasmid pKHNgAgo-F-GlnR. After the plasmid was transformed into M. smegmatis through electroporation for 3‒4 days, individual colonies were transferred to liquid LB media for further cultivation, and PCR was performed to confirm that the plasmids were successfully transferred into M. smegmatis (Fig. 3B). After three passages, PCR was performed, and the products were sequenced to further confirm that the glnR gene was indeed knocked out in strains with successfully transferred plasmids (Fig. 3A and C). Additionally, we extracted RNA from the WT and ΔglnR strains and conducted qRT‒PCR analysis. Our results revealed that in the ΔglnR mutant, the expression of glnR was absent, which indicated that glnR was knocked out successfully and that the NgAgo-F system can be used for genome editing in M. smegmatis (Fig. 3D and E).

To test whether NgAgo-F-based gene knockout in M. smegmatis could be applied to genes other than glnR, we applied this approach to manipulate another gene, ltmA. We recombined the 800 bp upstream and downstream homology arms of the ltmA gene and sfGFP fragment into the pKHNgAgo-F vector, obtaining the plasmid pKHNgAgo-F-ltmA. The plasmid was then transferred into M. smegmatis, and single colonies were selected for cultivation. Through fluorescence signals, we identified the strains that were successfully transformed with the plasmid pKHNgAgo-F-ltmA (Fig. 4B). After three passages, we confirmed the deletion of ltmA via PCR amplification with the primers ltmA-F/R and ltmA-R/gfp-R and obtained the ΔltmA mutant (Fig. 4A and C). To further substantiate the knockout results, RNA was extracted, and qRT-PCR analysis revealed that the expression of ltmA was eliminated in the ΔltmA mutant (Fig. 4D). These results further support that the NgAgo-F system can be used for genome editing in M. smegmatis.

Plasmid transformation is a necessary step for gene editing. The larger the plasmid is, the lower the transformation efficiency. For some difficult-to-transform strains, a small plasmid is considered essential for successful transformation. To further screen for small plasmids for NgAgo-F-mediated gene editing in M. smegmatis, we explored the impact of homology arm length on NgAgo-F cleavage activity in M. smegmatis, using glnR as the target. We constructed glnR gene knockout plasmids with homology arm lengths of 2 × 100 bp, 2 × 200 bp, 2 × 400 bp, 2 × 600 bp, and 2 × 800 bp, and separately transformed them into M. smegmatis to calculate the gene knockout efficiency (Fig. 5). The results indicated that the construction with a 2 × 100 bp homology arm length could not completely knock out the gene; a 20% knockout efficiency was achieved with a 2 × 200 bp homology arm length; a 60% knockout efficiency was achieved with a 2 × 400 bp homology arm length; an 80% knockout efficiency was achieved with a 2 × 600 bp homology arm length; and an 80% knockout efficiency was achieved with a 2 × 800 bp homology arm length (Fig. 5). Through comparison, we found a 600 bp homology arm length is sufficient to achieve efficient gene knockout, and that the minimum length of the knockout plasmid could be reduced to 200 bp. Reducing the length of the plasmid increased the likelihood of successful plasmid transformation into mycobacteria, leading to successful knockout of the target gene.

E. coli DH5α served as the host for plasmid construction and was used to assess NgAgo-F-assisted gene editing efficiency. E. coli BL21(DE3) was used for protein expression and purification. Additionally, M. smegmatis MC² 155 served as the host for evaluating NgAgo-F-assisted gene editing. Unless otherwise stated, E. coli DH5α, BL21(DE3), and M. smegmatis were grown in LB media at 37°C and 220 rpm, with appropriate antibiotics added to the broth as needed.

The NgAgo-F gene was synthesized by Sangon Biotech Co., Ltd. (Shanghai, China), with codon optimization. To construct the pKHNgAgo-F plasmid, the restriction enzymes EcoR I and EcoR V were used to obtain the pKC1139 vector. The Flag-NgAgo-F fragment and Hsp60 promoter were amplified via the primers Flag-NgAgo-F/R and Hsp60-F/F1/R. These two fragments have 25 bp overlapping sequences, and the two fragments were subsequently fused through PCR amplification. The fused fragment was connected to the pKC1139 vector via the pEASY-Basic Seamless Cloning and Assembly Kit (Transgene, Beijing, China). The upstream and downstream homology arms of the glnR gene and ltmA gene were amplified using M. smegmatis genomic DNA as the template. These homology arms were separately cloned and inserted into the pKHNgAgo-F vector, generating the plasmids pKHNgAgo-F-glnR and pKHNgAgo-F-ltmA. Finally, pKHNgAgo-F-glnR and pKHNgAgo-F-ltmA were transformed into M. smegmatis for further applications.

M. smegmatis was cultured in LB liquid media supplemented with 0.05% Tween 80 at 37°C for 2 days. Subsequently, 500 µL of the culture was added to 100 mL of LB liquid media containing 0.05% Tween 80 in a 500-mL shake flask and incubated at 37°C with shaking at 220 rpm for approximately 36 h. When the OD₆₀₀ reached 0.6, the cells were immediately placed on ice and incubated for 30 min. The cells were harvested and collected by centrifugation at 6,000×g at 4°C for 10 min. After the supernatants were removed, the cells were washed once with cold water and washed twice with cold 10% glycerol. Finally, the cells were resuspended in 500 µL of 10% glycerol, and 200 µL aliquots were divided into 1.5 mL sterile centrifuge tubes.

For electroporation, 20 µL of plasmid DNA was added to 200 µL of competent cells, and the mixture was transferred into precooled 2-mm electroporation cuvettes. Electroporation was performed at 3 kV for 4 ms once or twice via a MicroPulser (Bio-Rad, 411BR7769). Immediately, 900 µL of LB medium was added to the competent cells, and the mixture was incubated with shaking at 30°C and 220 rpm for approximately 3 h. Following recovery, the cells were centrifuged, and the cells in the final 200 µL were resuspended and plated on LB agar plates containing 50 µg/µL apramycin. The plates were incubated for 3‒4 days. To ensure transformation efficiency, competent cells should be prepared and used immediately.

The primers 28a-NgAgo-F-F/R were used to amplify the NgAgo-F fragment, and the primers pET28a-F/R were used to amplify the pET28a vector fragment. The two fragments had 25 bp overlapping sequences, and the overlapping sequences were designed with the primers pET28a-F/R. In the pEASY-Basic Seamless Cloning and Assembly Kit, a basic mixture was used. The assembly system contained 6 µL of Basic Mix, x µL of NgAgo-F fragment, and (6−x) µL of pET28a vector, and the mixture was incubated at 50°C in a water bath for 30 min. The molar ratio of the NgAgo-F fragment to the pET28a vector was 3:1‒5:1. The mixture was subsequently transformed into competent cells. T7 and T7 Ter primers were used to identify the correct clones. The recombinant plasmid pET28a-NgAgo-F was transformed into E. coli BL21(DE3) competent cells for protein expression. The transformed cells were initially cultured in LB liquid media at 37°C overnight. Then, 5 mL was added to 100 mL of LB liquid media in a 500 mL shake flask and cultured at 37°C with shaking at 220 rpm for approximately 2‒3 h. When the OD₆₀₀ reached 0.6, His-NgAgo-F was induced by adding 0.5 mM IPTG, followed by incubation at 18°C for 14‒16 h. The cells were collected by centrifugation at 6,000×g for 10 min and washed once with 1× PBS. After being resuspended in 35 mL of 1× PBS, the cells were lysed via sonication, and the supernatants were collected via centrifugation at 9,000×g for 20 min at 4°C. Protein purification was performed according to previously established protocols (19). The purified protein was analyzed by 15% SDS‒PAGE (Epizyme Biotech, Shanghai, China).

pKHNgAgo-F::M. smegmatis and M. smegmatis were cultured in LB liquid media supplemented with 0.05% Tween 80 at 30°C for 2 days. The cells were collected by centrifugation at 6,000×g for 20 min and washed once with 1× PBS. The cell pellet was resuspended in 300 µL of 1× PBS and lysed by sonication, and the supernatants were collected by centrifugation at 9,000×g for 20 min at 4°C. The supernatants of pKHNgAgo-F::M. smegmatis and M. smegmatis were mixed with 6× protein loading buffer and run on SDS‒PAGE gels. Next, the proteins on the SDS‒PAGE gels were transferred to a PVDF membrane (Merck, Millipore) at 300 V and 380 mA for 90 min. After being washed twice with TBST buffer, which consisted of 20 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 0.1% (v/v) Tween 20, the PVDF membrane was incubated with 0.5% BSA blocking buffer at 4°C overnight. Two microliters of anti-Flag antibody (Transgene, Beijing, China) was added to the BSA blocking buffer and incubated at 25°C for 1.5 h. Then, the PVDF membrane was washed three times with TBST buffer, and 2 µL ProteinFind Goat Anti-Mouse IgG (H + L) (Transgene, Beijing, China) was added to 10 mL of TBST buffer. After incubation for approximately 1 h, the PVDF membrane was washed three times with TBST, and an enhanced chemiluminescence (ECL) system (CTB, USA) was used to detect the signal.

WT, ΔglnR, and ΔltmA were cultured in LB media supplemented with 0.05% Tween 80 at 37°C for 2 days. Total RNA was extracted from WT, ΔglnR, and ΔltmA strains with an RNAprep Pure Cell/Bacteria Kit (Tiangen Biotech, Beijing, China) according to the manufacturer's instructions. cDNA was subsequently synthesized via TransScript Uni All-in-One First-Strand cDNA Synthesis SuperMix (Transgene, Beijing, China) for qRT‒PCR. A 96-well reaction was performed with a Bio-Rad CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA, USA) using TB Green Premix Ex Taq (TaKaRa Biotechnology, Beijing, China). All PCR mixtures were prepared in a volume of 20 µL in triplicate and contained 10 µL of SYBR Green PCR master mix, 0.4 µM forward primer, and 0.4 µM reverse primer. The PCR conditions were as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 30 s, 55°C for 20 s, 72°C for 15 s, and 72°C for 10 min. The expression levels of glnR and ltmA were normalized to that of the housekeeping gene sigA (MSMEG_2758).