Abuse of antibiotics and spread of antibiotic resistance genes has become concerns in multiple fields. To detect both antibiotics and their resistance genes accurately and fast, we came up with our detection system, CESAR. This is the very first system which applies Cas12a as an efficient tool for antibiotic resistance genes detection and can also detect antibiotics as well. To verify the system, proper target sequences was chosen and transferred into microbes as imitation of actual samples. We successfully extracted Cas12a from E. coli and synthesis needed crRNA for this system. To further ease the procedure for our future clients, four special designed modules (RPA-amplification module, aptamer-sensing module, Cas12a-reporting module and fluorescence-measuring module) that simplifies exam steps were created and will be verified here by experiments, modeling and literature information. With appropriate storage, CESAR can provide precise and rapid detection in a long time.
To verify our CESAR system, we have designed standard detection cassetten to simulate our target of examination. We selected potential target genes from most important ARGs (MCR-1 and NDM). The sequences, which is highly conserved in all species, are screened out through ClustalOmega. For better adaptation to our system, the online tool CRISPRdt is used to select protospacers with highest rankings for Cas12a. Best performance protospacers’ sequences will become our chosen target DNAs. We constructed them into vector through Zero TOPO-Blunt Cloning kit (YEASEN, China, #10909ES20). All target DNAs have been introduced with four restriction site and a “stop anticodon” on upstream for programmability and safety
Figure1. target sequence with restriction site and “stop anticodon”
The insertions are confirmed through sequencings
Figure2. sequencing of MCR-1 target A/B/C
The After inserted plasmid into E. coli (DH5alpha), we managed to mimic actual detection condition. We acquired 3 targets that comes from MCR-1 and one from NDM.
In this part, our team tested the validation of RPA for amplification of our target DNA. RPA is suitable for our design for its ability of thermostatic amplification. However, design of primers may highly influence the RPA efficiency. Thus, we designed different forward and reverse primers for each target and attempted to find out the best pair of primers which has highest amplification yield. All candidates were designed is based on previous experiences, (see detail in RPA part) and they were all prosperously constructed back to back into vector pESI – Blunt Simple vector (YEASEN, China, #10909ES20) for characterization of each pair of primers
Figure3. RPA primers being constructed back to back on vectors
This is a way that we came up with to quantify the efficiency of RPA primers. In the same condition, the amplification yield can be indicated by products’ concentrations and accurately reflect productivity of each pair of primers.
Through quantification of outcomes, we find out that MCR-1’s F3/R1 primer pair, which provided the highest yield, performed best after 20min reaction and NDM’s best primer pair F1/R3 are suitable for 60min reaction. Agarose gel electrophoresis results further insured our products are, indeed, target DNAs.
Figure4. The concentration of RPA products, being quantified by nanodrop at absorption of 260nm
Figure5. RPA products (left) and PCR products’(right) agarose gel electrophoresis results,
Cas12a detection was also applied on RPA products. It has shown that, with the same amount of RPA and PCR products, RPA gave great performance: high intensity of fluorescence.
Figure6. Cas12a detection results of PCR product and RPA product with same amount.
This indicates that out RPA is very efficient if it was used in clinic or other conditions.
One key element of our system is the enzyme, AsCas12a. With skilled operation, we extracted and purified it by ourselves. Our target protein’s expression vector was obtained from Yunjie Xiao from Tianjin University. This vector was further designed with MBP-his tag1. The his-tag allowed us to purify protein with Ni-NTA beads, meanwhile MBP-tag was responsible for increasing expression of Cas12a and help is fold and dissolve. We gained AsCas12a after culturing, collection, crushing, and affinity purification. It was assured by SDS PAGE gel, which result matched AsCas12a .
Figure7. The result of SDS PAGE indicates the extracted protein is around 130kD, which matches AsCas12a
Another key element is crRNA. We used T7 in vitro reverse transcription kit (NEB HiScribe T7 Quick High Yield RNA Synthesis Kit) to synthesis crRNA. By combining our designed RNA sequence (RNA scaffold and with designed target DNAs as spacers) with T7 promoter, we can easily get crRNA for future usage.
Figure8. crRNA templet for reverse transcription, which have T7 protomer, RNA scaffold and spacer (target sequence)
With Cas12a, crRNA and target, our system can now be functional with only one element left, a reporter that can be trans-cleaved. We purchased a short single-strand DNA (ssDNA) with fluorescence group FAM at one end and a quenching group BHQ1 at the other end. The sequence of this probe is 5’-TTATT-3’, which was designed with help of Dr. Huang, who is an expert of CRISPR/Cas system.
At first , Cas12a was incubated with designed crRNA, the Cas12a would assemble with crRNA. After forming a complex, Cas12a will turn to a relatively stable conformation. In this form, we could store the complex as the material for the next reaction.
To quantify the intensity of fluorescence in microplate reader, we diluted this system to 100ul solution with final concertation of each elements become: Cas12a, 200nM; crRNA, 250nM, target DNA, 40nM and reporter ssDNA, 50nM. The system can be concentrated if our product can finally go into commercial use. In this system, the fluorescence reached to a high level in about 20min, which can be visualized by naked eye, and it did not fall in the next 8 hours
Figure9. The green fluorescence can be observed by naked eyes under blue light
Figure10. The fluorescence intensity was measured by microplate reader, the intensity level reach to a high level in a short time.
This suggested our system can provide accurate detection in a short time and can also preserve its signal in a long time if needed. In addition, our system also has the potential to quantify target DNA.
Besides detection of antibiotic resistant genes, our system can identify antibiotics as well. This module was designed for this usage in CESAR-I. We create a new element, Aptamer-Sandwich, (fig11) with three DNA strands, two Kanamycin aptamer and a ssDNA called activator. The activator was designed as the target for Cas12a recognition, which will be paired and locked by the two modified aptamers before using. When Kanamycin existing, the aptamers will release activator. And the activator will then activate Cas12a, so that we can detect antibiotics by this part (details are described in Engineering Success ).
Figure11. Aptamer-Sandwich desgin
We used this system to verify antibiotic detection efficiency of CESAR-I. We did orthogonal experiments (table1&2) concerning the concentration of Aptamer-Sandwich and kanamycin. We found out that fluorescence strength has positive relation with Kanamycin concentration (fig12&13). The additional discover was that, as Sandwich’s concentration rise, the complex have relatively low fluorescence intensity during experiment (fig13), this may be due to excessive activator may suppress reaction due to unbalance ratio. This suggests that appropriate Aptamer Sandwich concentration is important. The reason remains unknown and we plan to design more examination for different concentration of target sequence to verify our hypothesis. Despite of that, the result proposed we could not only detect, but also quantify antibiotic residue in environment.
Figure12. Absorption of different concentration Kana or Aptamer-Sandwich are shown in picture. Each number’s concentration are shown in Table.1
Table1 & 2: orthogonal experiments’ condition Sandwich=Aptamer-Sandwich
Figure13. The result of orthogonal experiment
When testing the whole system, we found that reaction happening in one pot will dramatically decrease the detection efficiency for the samples in RPA-amplification step or aptamer-sensing step will be disturbed by Cas12a cleavage. To solve this problem, we invented a novel method to separate and automatically deliver reaction materials in Cas12a-reporting module into the pre-processed samples. In our design, the materials for Cas12a reporting will be held on a liquid membrane on an iron ring. The iron ring will be previously stuck on the lid by electromagnet attraction. And it will fall down into the tube when electromagnet power off automatically.
This design is creative and suitable for our device. But how much liquid can it carry? One modeling was done to affirm the viability of this layout. Based on surface energy equation we constructed our modeling, called Catenoid Surface Model (CSM) for us to simulate and calculate. The result shows that we can deliver up to 3ul binary complex solution on a ring of radius 5mm, which is much more than we need. This model proved the feasibility of our design of iron ring. (refer to Modeling part 3 for details)
Storage of enzyme and RNA is important. Although crRNA and Cas12a’s complex can stabilize them, the proteinase and RNase can both causes impair of their function. Storage in low temperature condition will resourcefully slow this process. To prevent ice crystal and alternate freezing and thawing effect proteins’ conformation, we stored pre-incubated Cas12a and crRNA’s complex with 50% glycerol at -20°C condition distribute to one portion’s amount each. In that case, the complex should reserve its function for 1 year or more2
After hardworking, we ensured all modules can function well in whole experiment. Combined all parts, target sequence, RPA, Cas12a crRNA complex, aptamer, and whole device design, we successfully accomplished our intention, to detect antibiotics and resistance genes by one simple kit. In addition, we made effort on commercializing our system CESAR. We tried our best to mimic actual usage and simplify using procedures, to make sure CESAR could be used in real application scenario.
1 Nallamsetty, Sreedevi, Brian P Austin, Kerri J Penrose, and David S Waugh. 2005. “Gateway Vectors for the Production of Combinatorially-Tagged His 6 -MBP Fusion Proteins in the Cytoplasm and Periplasm of Escherichia Coli.” : 2964–71.
2 Nakamura, Hajime et al. 1998. “Measurements of Plasma Glutaredoxin and Thioredoxin in Healthy Volunteers and during Open-Heart Surgery.” Free Radical Biology and Medicine 24(7–8): 1176–86.