1. 1ST ITERATION
Optimization of CRISPR-MAD7(Cas12a) system
1.1 1st ITERATION – design
For gene editing using CRISPR technology, you need to transfect the plasmid into cells, allow it to express the CRISPR system and edit the target gene. To improve the efficiency of gene editing, we optimized the CRISPR system to determine the optimal CRISPR-MAD7(Cas12a) system.
The promoter is responsible for driving the transcription of genes, which increases the localization and binding of CRISPR system on the target genes, thus improving the editing efficiency is the basic structure of plasmid. In order to effectively deliver CRISPR components to the target cells, the most appropriate plasmid should be selected. In CRISPR system, Cas protein is usually improved by identifying and binding to the PAM site and performing DNA cutting or other modifications to identify the recognition efficiency of Cas protein by selecting the best PAM site.
In conclusion, we will select the optimal promoter, plasmid and PAM site to improve the efficiency of gene editing.
1.2 1st ITERATION – build
A. Selection of the optimal promoter
To determine the optimal promoter, we went through literature review and screening, the relevant promoters were Ptuf, PlacM, Ptac and Ptrc. The insertion of these promoters into the pEC-XK99E plasmid vector resulted in the recombinant plasmid. The constructed plasmids were transferred into E. coli and then cultured. After some time, colonies on the medium were picked for PCR validation, an expanded culture of the verified verified bacteria. After 12h, the plasmids were extracted and sequenced, electrotransfer of the correctly sequenced plasmid into C. glutamicum ATCC13032. Colony growth on the medium was observed after a certain period of incubation. The optimal promoter was identified by alignment.
B. Selection of the optimal plasmid
We found that the gene editing efficiency of the pEC-XK99E plasmid vector that identified the best promoter was still not high enough, and to improve the efficiency, we selected the best plasmid. We constructed pJYS1-MAD7-TTTA, pBluescript-MAD7-TTTA and pXMJ19-MAD7-TTTA three plasmids. The correctly sequenced plasmid was electrotransferred into C. glutamicum ATCC 13032, and the colony growth on the medium was observed, and the optimal plasmid was determined by comparison.
C. Validation of promoters on the optimal plasmid
To ensure that promoters capable of efficient expression on the pEC-XK99E plasmid are also highly expressed on the newly determined optimal plasmid, validations of the promoter on the optimal plasmid was performed. The experimental manipulation of the first step was repeated after replacing the plasmid growth of the pEC-XK99E plasmid with the best plasmid.
D. Selection of the best PAM site
In the above experiments, we identified the optimal promoter and the optimal plasmid, based on which we will determine the optimal PAM site. By reviewing the literature, we selected (Table 1) PAM sites for experiments. Changing the gRNA during plasmid construction, that is, the PAM site was changed. The recombinant plasmid was obtained by fusion PCR and one-step cloning technology, the sequenced plasmid was electrotransferred to C. glutamicum ATCC13032, the colony growth was observed, and the best PAM site was obtained by comparison.
| Selection of the PAM sites |
|---|
| pJYS1-MAD7-gRNA-TTTA(+) |
| pJYS1-MAD7-gRNA-TTTC(-) |
| pJYS1-MAD7-gRNA-TTTT(-) |
| pJYS1-MAD7-gRNA-TTTG(-) |
| pJYS1-MAD7-gRNA-TTTC(+) |
| pJYS1-MAD7-gRNA-TTTA(-) |
| pJYS1-MAD7-gRNA-CTTC(+) |
| pJYS1-MAD7-gRNA-CTTA(+) |
| pJYS1-MAD7-gRNA-CTTG(-) |
| pJYS1-MAD7-gRNA-CTTT(+) |
1.3 1st ITERATION – test
We initially estimated the successful introduction of the plasmid into the colonies on the plate, and verified it by colony PCR. For successful validation, the solution after colony PCR reaction was taken for gene sequencing to test whether successful knockout of related genes.
Selection of the optimal promoter
By contrast, we can obtain the optimal promoter that is PlacM.
By contrast, we can obtain the optimal plasmid that is pJYS1.
The comparison showed that the best promoter PlacM selected when using plasmid pEC-XK99E was also the most efficient knockout gene in plasmid pJYS1.
- Selection of the best PAM site
Since there are many PAM sites and many plate maps, we selected two representative plate plots here.
By contrast, we can obtain the optimal PAM site that is TTTC(+).
1.4 1st ITERATION – learn
We obtained the gene editing efficiency by comparing and calculating the color of the colonies on the plate, and selected the best parts.
The best promoter is PlacM. The best plasmid is pJYS1. The best PAM site is TTTC(+).
In summary, we have optimized the CRISPR system to obtain the pJYS1-MAD7-gRNA-TTTC(+) plasmid, and we will expand the subsequent experiments accordingly.
2. 2ND ITERATION
Build strains that produce high lycopene
2.1 2nd ITERATION – design
In the previous research, we have optimized the CRISPR-MAD7(Cas12a) system to improve the efficiency of gene editing of the CRISPR-MAD7 system in C. glutamicum ATCC13032 by selecting the plasmid, optimal PAM site, promoter, etc.Therefore, by using the optimized CRISPR-MAD7(Cas12a) system to edit the lycopene-producing gene of C. glutamicum ATCC13032, the purpose of lycopene enrichment can be achieved.
We first build the relevant plasmids of the CRISPR-MAD7(Cas12a) system that have been optimized in the early stage. And designed to accurately locate the gene crtEb, crtR upstream and downstream gRNA. Under the action of this gRNA, the downstream genes crtEb, crtR of lycopene can be accurately knocked out, so as to achieve the accumulation of lycopene.
2.2 2nd ITERATION – build
- pJYS1-MAD7-ΔcrtEb(Knock out the crtEb gene)
- pJYS1-MAD7-ΔcrtR(Knock out the crtR gene)
- pJYS1-MAD7-ΔcrtEb:ΔcrtR(Knock out the crtEb/crtR dual gene)
A. The plasmid was transferred into Escherichia coli Top10 competent cells by heat shock and cultured in LB solid medium at 37°C.
B. Verify and extract plasmid.Through resistance screening, colony PCR verifies whether the plasmid is successfully introduced into Escherichia coli Top10. For the successful verification of bacteria, the bacteria were expanded and cultured, and the plasmid DNA was extracted the next day and sent to the company for gene sequencing.
C. Transfer the sequenced correct plasmid into C. glutamicum ATCC13032 by electroporation and culture in LB solid medium at 30°C.
2.3 2nd ITERATION – test
First, through the naked eye, we can find that the color of the colony has changed, and the plasmid introduction is preliminarily estimated to be successful, and the colony PCR verification is performed. For successful verification of bacteria, the solution after colony PCR reaction was taken for gene sequencing to verify whether the relevant genes were successfully knocked out.
For cultures that verified successful knockout genes, we transferred them to 300 mL flasks for scale-up. After a certain period of time, a certain sample bacterial solution was taken and the yield of lycopene in the modified strain was measured by high performance liquid chromatography (HPLC) to evaluate the effect of experimental manipulation on lycopene synthesis.
2nd ITERATION – learn
In the previous project, we optimised the CRISPR-MAD7(Cas12a) system to achieve the highest gene editing efficiency in Corynebacterium glutamicum, and thus we wished to construct strains with a high lycopene yield that could produce lycopene in large quantities. We gradually constructed ΔcrtEb, ΔcrtR, ΔcrtEb:ΔcrtRstrains. Through the analysis we can find that knocking out the branched genes can lead to a good increase in lycopene yield. It helps us to better solve many problems, such as low efficiency of lycopene in industrial production.
