The DBTL cycle in engineering design is the core operating mode of synthetic biology. This project achieved DBTL cycling through three parts: the design and construction of a three plasmid system, the mining and testing of sacBs with different toxicities, and the validation of high-precision MAD7 modification. These works have enabled us to achieve ideal products through successful engineering processes. The team has gained a deeper understanding of synthetic biology from multiple engineering experiences.
Based on the principle of CRISPR-Cas system and various screening gene characteristics, we have designed a three plasmid system that can achieve high-throughput, accurate screening of MAD7 mutations. The screening system is designed and constructed based on pBBR1MCS-2 and P15A plasmids, incorporating on-target and off-target sequences, and selecting sacB gene and ampicillin (Amp) resistance gene as the selection markers. We will also introduce MAD7 gene and gRNA expression sequence into pSC101 plasmid, and introduce temperature-regulated promoter andChloramphenicol resistance gene for transcription control and characterization. Ultimately, we will construct a new three-plasmid screening system, which will be named EP1-on, P15A-off, and EP7-wt (Figure 1).
When screening, we need to control the temperature to induce the expression of MAD7 nuclease in Escherichia coli. When MAD7 nuclease specifically cleaves the on-target sequence, the sacB gene cannot be expressed. On the other hand, if the MAD7 nuclease does not cleave the off-target sequence without off-target, then the AmpR gene is expressed normally. Ultimately, we can obtain high-precision MAD7 nucleases with low off-target rates in Escherichia coli grown normally on Chl+Amp+Sucrose medium.
Based on the plasmid structure, we first used methods such as gibson recombination and CPEC to ligate the MAD7 gene, sacB gene, and antibiotic resistance gene into the plasmid. Then, based on the HBsAg gene, we determined on-target and off-target. The construction of a three plasmid screening system was completed by introducing on-target and off-target sequences into the target plasmid through SDM.
After plasmid construction, we validated the screening function of the three plasmid system using MAD7 (WT).
EP1-on validation: Three plasmid systems were electroporated and introduced respectively into two E.coli K-12 competent cells containing EP1 plasmid. After recovery, the cells were plated onto chloramphenicol (Chl) + Ampicilin (Amp) + Sucrose,and Chl + Amp culture media using drop inoculation method, respectively.
P15A-off validation: the three plasmid systems were electroporated and introduced into two E.coli K-12 competent cells containing EP1. After recovery, they will be plated ontoChl + Amp + Sucrose andChl + Sucrose culture media using the dilution drop method respectively.
Due to the lethal effects of sacB and Amp, the CFU grown normally in the experimental group was significantly different from that in the control group. (Figure 2)The results of DNA sequencing and validation experiments indicated that we have successfully constructed a three plasmid system containing EP1-on, EP7-wt, and P15A-off, and the system performed its screening function normally.
Through the attempt to construct a three plasmid system, we have gained an understanding of the mechanisms of action of relevant components, proteins, and DNA sequences. After experimentation and discussion, the team members raised doubts about the screening effectiveness of the three plasmid system. Different concentrations of antibiotics and sucrose conditions may affect the screening effect. Inappropriate toxicity of sacB may also lead to unsatisfactory screening results. Therefore, we explored these issues and identified two different toxicity sacB mutants.
The sacB encoded fructosyltransferase can catalyze the hydrolysis of sucrose and the synthesis of fructooligosaccharides, and the accumulation of fructooligosaccharides can lead to death in Escherichia coli. In order to develop a more flexible screening toolkit for sacB compositions with different lethal abilities, we predicted single and combined mutation sites that affect sacB toxicity through molecular docking simulations and Funclib. Based on the prediction results of different technologies, we propose sacB-S164A and sacB_E262Q mutants with different toxicities, and expect to verify their toxicity through wet experiments.
Based on the different toxicity of sacB mutations predicted by the dry experiments, we designed primers for two mutations in the sacB gene, S164A and E262Q, and constructed EP1-on-sacB_S164A and EP1-on-sacB_E262Q plasmids. Through electroporation, expansion culture, plasmid extraction, and DNA sequencing, we successfully obtained two types of EP1-on plasmids carrying sacB_S164A and sacB_E262Q mutants, respectively.
We transformed the aforementioned two EP1-on plasmids into competent E.coli K-12 cells, and dropped onto Kanamycin (Kan) and Kan + 0.1% Sucrose media, respectively after recovery, with sacB_WT and sacB_S164T serving as the controls. Upon calculation, the lethality rates of sacB-S164A and sacB_E262Q were 19.33% and 41.35%, respectively.
By comparing the toxicity of the above two mutants with sacB_S164T and sacB_SWT proposed by the DUT China team in 2022, we have enriched the sacB toolkit covering different mortality rates. (Figure 3) The existing three types of sacB mutations all show a decrease in mortality rate, and we have not been able to successfully predict the mutation site that increases the mortality rate of sacB. In the future, we will try different prediction methods to obtain more sacB mutants.
In order to solve the safety problem of off-target effect in gene editing treatment of hepatitis B, we hope to develop a high-precision MAD7 nuclease. Using zero-shot mutational effect prediction and error-prone PCR, we hope to obtain highly accurate and highly active MAD7 mutants for gene editing therapy.
We transformed the aforementioned two EP1-on plasmids into competent E.coli K-12 cells, and dropped onto Kanamycin (Kan) and Kan + 0.1% Sucrose media, respectively after recovery, with sacB_WT and sacB_S164T serving as the controls. Upon calculation, the lethality rates of sacB_S164A and sacB_E262Q were 19.33% and 41.35%, respectively.
After obtaining the MAD7 mutation library, we need to ligate the MAD7 mutation fragments with the EP7 plasmid. First, using the wild-type EP7 plasmid as a template, we obtained a large number of backbone fragments through PCR amplification (Figure 6). Subsequently, after gel electrophoresis, Dpn I digestion, etc., the backbone fragments were extracted and purified. Then, we ligated the MAD7 mutation fragments with the backbone through gibson assembly. Finally, through electroporation, plating, scraping, plasmid extraction, etc., we collected all EP7-mutant plasmids.
We introduced the EP7-mutant plasmid into E.coli K-12 competent cells containing EP1-on and P15A-off via electroporation, establishing a three plasmid screening system. After recovery, the E.coli cells were dropped onto a plate containingChl + Amp+ 0.1% Sucrose, and also onto aChl plate as a negative control. MAD7(WT) was used as a positive control (Figure 7). Finally, we obtained 14 and 24 single colonies at concentrations of 10-2 and 10-1 using the drop inoculation method. The off-target rate of the MAD7 mutant included may be significantly lower than that of MAD7 (WT).
After spreaking, 7 out of the 38 single colonies selected above could not grow onChl + Amp plate, indicating that the MAD7 mutant had undergone off-target cleavage. Therefore, we eliminated these 7 mutants.
The E.coli strain obtained through screening still contained two plasmids, EP7-mutant and P15A-off. We used serial passaging and gel extraction methods to remove the P15A plasmid from the E.coli cells, obtaining the EP7 single plasmid containing the mutant fragment. To calculate the editing efficiency of each MAD7 mutant, E.coli K-12 cells containing EP7-mutants and EP1-on plasmids were dropped onto Sucrose + Chl andChl media, with MAD7(WT) serving as a control. To calculate the off-target rate of each MAD7 mutant, E.coli K-12 cells containing EP7-mutants and P15A-off plasmids were dropped onto Amp +Chl andChl media.
After calculating the editing efficiency and off-target rates (Figure 8, 9), we discovered several mutants in the high-precision MAD7 mutants obtained through screening that can significantly reduce the off-target rate while ensuring the high editing efficiency. Among them, the mutant No. 6 has an off-target rate of 3.17% when both on-target and off-target existed. Compared to the off-target rate of 99.02% for MAD7(WT), the off-target rate of the mutant No. 6 has decreased by 96.8%, and the editing efficiency is 59.51% of MAD7(WT). This mutant MAD7 is named HF-MAD7. HF-MAD7, with its low off-target rate and stable editing efficiency when editing the HBsAg gene target, may become a reliable tool for gene editing therapy of hepatitis B.
This mutant MAD7 is named HF-MAD7. HF-MAD7, with its low off-target rate and stable editing efficiency when editing the HBsAg gene target, may become a reliable tool for gene editing therapy of hepatitis B. The acquisition of high-precision MAD7 mutants validates the success of this engineering design. However, the team found that after screening for E.coli, there may be both EP7-mutant and P15A-off plasmids present. This will cause inconvenience in quickly obtaining MAD7 mutations. Therefore, we hope to further optimize the design to overcome this problem.
