Sensitive detection of a bacterial pathogen using allosteric probe-initiated catalysis and CRISPR-Cas13a amplification reaction

The ability to detect low numbers of microbial cells in food and clinical samples is highly valuable but remains a challenge. Here we present a detection system (called ‘APC-Cas’) that can detect very low numbers of a bacterial pathogen without isolation, using a three-stage amplification to generate powerful fluorescence signals. APC-Cas involves a combination of nucleic acid-based allosteric probes and CRISPR-Cas13a components. It can selectively and sensitively quantify Salmonella Enteritidis cells (from 1 to 105 CFU) in various types of samples such as milk, showing similar or higher sensitivity and accuracy compared with conventional real-time PCR. Furthermore, APC-Cas can identify low numbers of S. Enteritidis cells in mouse serum, distinguishing mice with early- and late-stage infection from uninfected mice. Our method may have potential clinical applications for early diagnosis of pathogens.


1-The manuscript by Shen et al describes a new method for microbial detection.
The method is based on the combination of two known techniques: allosteric DNA probes and CRISPR-Cas enzymes. The Allosteric DNA probes have already been used for detection, and the authors here combine these probes with a CRISPR-Cas system with the purpose of adding two levels of signal amplification through DNA replication and transcription to improve the detection limit. This combination is novel and has not been reported before, and to my knowledge, this is the first use of CRISPR techniques for microbial detection. The manuscript is well-written and technically sound.

2-I have two main concerns:
A-The problem with aptamers: The efficiency of using aptamers for large targets such as microorganisms. Despite the abundant literature in the field, there is no commercial detection assay using aptamers as receptors because of their lack of specificity and poor reproducibility. The only commercial use of aptamers today is for therapeutic reasons and on very small targets (molecules). Given these concerns, the authors should at least provide more information on the aptamers used in this work. Are they commercially obtained, are they produced by the team? did they characterize the binding properties of the aptamers (affinity, dissociation constants)?
B-The problem with "single pathogen" detection: One of the major problems when we attempt to perform low detection limits with low number of cells is that the probability of having microorganisms in your diluted samples decreases exponentially. If we dilute 10 times a 1mL solution with 10 CFU, the result is rarely 1 CFU per mL. The chance to get 1 CFU per mL is very slim. As a result, one should analyze all the diluted samples to ensure a positive detection, and even then, it's hard to demonstrate that there is only one single pathogen your sample. I would suggest avoiding titles with "single pathogen" detection claims.
3-Cost of the assay: One of the main motivations of the authors is to offer a sensitive and affordable microbial detection assay. It is true that the developed assay overcomes the need for antibodies. However, this assay uses three different types of enzymes: DNA polymerase, RNA polymerase and CRISPR-Cas13a, and require equipment for DNA amplification, which would significantly increase the cost of the assay. It would be helpful if the authors could estimate the cost of the assay for a single test. 4-Time of detection: The proposed assay involves 5 biochemical steps: target recognition by the aptamers, DNA replication, RNA transcription, RNA recognition by the enzyme Cas13, and Cleavage of the reporter RNA by Cas13. It would be helpful if the authors estimate the time needed for each step and the overall time of the assay.
We appreciate all reviewers for their valuable comments and suggestions. Our co-authors have conducted a series of experiments to address the raised concerns in the past three months. Our response to review comments together with new experimental results is given in this letter and the revised manuscript (marked in green). In the following, we present our response (marked in blue) to each review comment (marked in yellow) in detail.

Reviewers' comments:
Reviewer #1 (Remarks to the Author): The study by Shen, et al. describes a new and innovative assay to detect a pathogen, such as Salmonella enteritidis contamination in food or body fluids. The assay utilizes an allosteric probe, which is composed of an aptamer sequence specific for the target bacteria (S. enteritidis). Although poor signal-to-noise limits the sensitivity of such detection methods, this assay utilizes a CRISPR-Cas13a step to amplify a fluorescence signal. In sum, the assay appears more sensitive and faster than conventional real-time PCR methods, suggesting its potential in applications involving disease diagnosis and food safety inspection.
The approach is innovative, but there are some limitations that might prevent it from becoming widely adopted.
1. There are insufficient details about the aptamer. What is its molecular target? Was it discovered against a particular protein, or against intact S. enteritidis? Did the authors discover it, or did someone else? If the latter, please cite accordingly. What are its binding kinetics?
Answer: Thanks for the concern on this issue. The target of aptamer is intact live S. enteritidis. The DNA sequence of aptamer domain in allosteric probe used in our paper was Clone SE-3 (60 nt) which was discovered by Kolovskaya   experiments among these five S. enteritidis strains were performed under the same experimental condition using APC-Cas (Supplementary Fig. 7). Supplementary Fig. 7A shows all five S. enteritidis strains give significant fluorescence increase at the level of 1×10 3 CFU within 30 min, comparing to the background with no S. enteritidis. In Supplementary Fig. 7B, The fluorescence growth rate (V g ) of all five S. enteritidis strains is over at least 12-fold higher than that of background, and the V g of those four S. enteritidis strains differs from 94% to 130% of the S. enteritidis strain (CMCC 50040) used in the manuscript. Based on these results, it seems the aptamer and assay is compatible for different S. enteritidis strains within the same serotype. We have added Supplementary Fig. 7 and associated description on page 15 and 16 in the revised manuscript.

Strains
Serovars Answer: We thank the reviewer for this concern. At present, some S. enteritidis strains appear to be insensitive to antibiotics in clinical practice. However, these strains carry different drug resistance genes, which leads to inconsistent drug resistance phenotypes.
Methicillin-resistant Staphylococcus aureus (MRSA), which is typically called as a "superbug" in the Staphylococcus aureus (SA) family, 2 is with a clear drug-resistant phenotype and a clinically common bacteria. So, MRSA was chosen as a model to study whether APC-Cas system could be applied for antibiotic resistant bacteria. We redesigned and synthesized a new allosteric probe with the aptamer domain specifically targeting MRSA. The DNA aptamer against MRSA was developed by Ocsoy et al. 3 The DNA sequences used in this experiment is provided below in Table L1. SA was used as control. The results in Fig. L1 showed that APC-Cas could truly distinguish MRSA from SA under the same amount of bacteria (1×10 3 CFU). The V g of MRSA was over 3.5-fold higher than that of SA. These new results demonstrate that APC-Cas could be applied for antibiotic resistant bacteria detection in the future.
Although these results show the possibility of antibiotic resistant bacteria detection of APC-Cas, we feel that the redesigned assay for testing MRSA is preliminary. Therefore, we decide that these results and associated information for MRSA test are just shown in rebuttal   Overall, the assay offers potential improvement over existing approaches, but is quite limited-specifically for only S. enteritidis. This study is appropriate for a specialized journal. we chose Clone SE-3 (60 nt) to be used in our APC-Cas system based on its sequence length, dissociation constant (K d ), bacteria growth suppression and other performances. We have added the reference and associated description on page 7 in the revised manuscript.

Answer
AP in APC-Cas system is a single-stranded DNA molecule, DNA molecules are susceptible to nuclease-mediated degradation. Unmodified DNA molecules are quickly degraded by nucleases in biological samples, especially in blood. 2,3 It is reported that 3' exonuclease is primarily responsible for the degradation of oligonucleotides in serum, and phosphorylation at the 3' end of DNA can render the DNA molecule resistant to enzymatic hydrolysis. 4 Therefore, a phosphate group was labeled at the 3' end of AP as mentioned in the manuscript. In addition, 3' phosphorylation can also prevent DNA polymerase from recognizing DNA ends and then inhibit extension reactions. We have added the references and associated description on page 5 in the revised manuscript.
two levels of signal amplification through DNA replication and transcription to improve the detection limit. This combination is novel and has not been reported before, and to my knowledge, this is the first use of CRISPR techniques for microbial detection. The manuscript is well-written and technically sound.

2-I have two main concerns:
A-The problem with aptamers: The efficiency of using aptamers for large targets such as microorganisms. Despite the abundant literature in the field, there is no commercial detection assay using aptamers as receptors because of their lack of specificity and poor reproducibility. The only commercial use of aptamers today is for therapeutic reasons and on very small targets (molecules). Given these concerns, the authors should at least provide more information on the aptamers used in this work. Are they commercially obtained, are they produced by the team? did they characterize the binding properties of the aptamers (affinity, dissociation constants)?
Answer: We thank the reviewer for this comment. Reviewer #1 and #2 also raised the similar concern. The DNA sequence of aptamer domain in allosteric probe (AP) used in this assay was discovered by Kolovskaya et al. 1  B-The problem with "single pathogen" detection: One of the major problems when we attempt to perform low detection limits with low number of cells is that the probability of having microorganisms in your diluted samples decreases exponentially. If we dilute 10 times a 1mL solution with 10 CFU, the result is rarely 1 CFU per mL. The chance to get 1 CFU per mL is very slim. As a result, one should analyze all the diluted samples to ensure a positive detection, and even then, it's hard to demonstrate that there is only one single pathogen your sample. I would suggest avoiding titles with "single pathogen" detection claims.
Answer: We agree with the reviewer's comment that limited dilution method is hard to guarantee there is only one single pathogen in the gradually diluted sample. As mentioned in our original manuscript on Page 12, "To verify the sensitivity of APC-Cas, 1, 3 and 8 S.
enteritidis cells were picked out and performed with APC-Cas, individually." We used micropipette to pick out 1, 3, 8 S. enteritidis cells to perform the experiments, and the picked amount of S. enteritidis was further quantified and demonstrated by the plate colony counting method (Supplementary Fig. 3). Therefore, we think single pathogen detection could be claimed in the title of our manuscript. Answer: Thank you for this suggestion. Although APC-Cas system contains three different types of enzymes: DNA polymerase, RNA polymerase and CRISPR-Cas13a, the total reaction volume is 10 µL and the tested sample volume is only 2.5 µL. Therefore, the amount of enzymes and other components used in this assay is quite small for a single test. As shown in