key: cord-303986-9g24xg9x
authors: Huang, W.; Yu, L.; Wen, D.; Wei, D.; Sun, Y.; Zhao, H.; Ye, Y.; Chen, W.; Zhu, Y.; Wang, L.; Wu, W.; Zhao, Q.; Xu, Y.; Gu, D.; Nie, G.; Zhu, D.; Guo, Z.; Ma, X.; Niu, L.; Huang, Y.; Liu, Y.; Peng, B.; Zhang, R.; Zhang, X.; Li, D.; Yang, G.; Liu, L.; Zhou, Y.; Wang, Y.; Hou, T.; Gao, Q.; Li, W.; Chen, S.; Hu, X.; Han, M.; Zheng, H.; Wen, J.; Cai, Z.; Song, F.; Zhao, G.; Wang, J.
title: A CRISPR-Cas12a-based specific enhancer for more sensitive detection of SARS-CoV-2 infection
date: 2020-06-05
journal: nan
DOI: 10.1101/2020.06.02.20119735
sha: 
doc_id: 303986
cord_uid: 9g24xg9x

High Ct-values falling in the grey zone are frequently encountered in SARS-CoV-2 detection by real-time reverse transcription PCR (rRT-PCR) and have brought urgent challenges in diagnosis of samples with low viral load. Based on the single-stranded DNA reporter trans-cleavage activity by Cas12a upon target DNA recognition, we create a Specific Enhancer for detection of PCR-amplified Nucleic Acids (SENA) to confirm SARS-CoV-2 detection through specifically targeting its rRT-PCR amplicons. SENA is highly sensitive, with its limit of detection being at least 2 copies/reaction lower than that of the corresponding rRT-PCR, and highly specific, which identifies both false-negative and false-positive cases in clinic applications. SENA provides effective confirmation for nucleic acid amplification-based molecular diagnosis, and may immediately eliminate the uncertainty problems of rRT-PCR in SARS-CoV-2 clinic detection.

The performance of SENA was quantitatively characterized via a systematic titration upon 111 rRT-PCR amplicons employing pure SARS-CoV-2 RNA standards comprised of the O and N 112 fragments as the templates. As it is aware that the viral nucleic acids extracted from patients' 113 samples such as nasopharyngeal swabs usually contain some biological and chemical contaminants 114 that might inhibit the enzyme activities for reverse transcription and PCR reactions (19) and is 115 likely one of the causal effects attributed to the low efficiency of rRT-PCR in clinical analysis. In 116 order to mimic the clinical sampling for the titration experimentation, the RNA standards were 117 serially diluted in buffer prepared by mixing the nucleic acid extracts from 40 COVID-19 negative 118 people, generating RNA templates ranging from 0.025 to 25 copies per reaction (Rx). 119 Due to the Poisson distribution property of sampling, replica variations become extremely 120 significant when the template copies in individual reaction are designed to be low, i.e., less than 121 3-4 copies/Rx, near the limit of detection (LoD) for rRT-PCR (20, 21), and extremely low, i.e., 122 equal to and less than 1 copy/Rx. To overcome this sampling ambiguity problem, we performed 9 123 replicas for groups with 1 and 0.5 RNA template copies/Rx while 6 replicas for each of the rest 124 concentrations. In addition, although the rRT-PCR assay supplier, Shanghai BioGerm (BJ), who 125 follows the Chinese CDC recommended primer sets (Table S1) decrease of the RNA templates to less than 3 copies/Rx, the rRT-PCR Ct values in some replicas, 131 primarily that corresponding to the N gene, passed 38 (the cut-off for positive as recommended by 132 the rRT-PCR kit suppliers) but were less than 40, which should be considered as entering the "grey 133 zone". The Ct values increased steadily when the concentration of the RNA templates further 134 decreased, with more and more replicas showing one or both Ct values entering the "grey zone" 135 and eventually all became "negative", i.e., greater than 40 or even 45 (Figure 2A , Table S2 and 136 Figure S1 ). Employing Ct=38 as the cut-off for "positive" detection, we estimated the LoD for O 137 and N genes with 95% confidence interval (CI) of this set of rRT-PCR assay as 3.3≤4.0≤6.1 and of the sample over that of the negative control at certain time point), we defined a parameter, 146 FCratio, which is the ratio of the FC at 10 min to that at 5 min after the initiation of fluorescence 6 some of the ambiguity readouts found with N-SENA ( Figure S1 and Table S2 ). Based on these 156 results, the mixFCratio was demonstrated as the most sure-proof index for rRT-PCR confirmation, 157 and we empirically estimated that mixFCratio≥1.145 for positive cut-off, and mixFCratio≤1.020 158 for negative cut-off (Figure 2A) . Of course, these two parameters are subject to further verification 159 and adjustment along with the increase of tested samples. Because SENA is rRT-PCR based, the 160 same methodology for determining the rRT-PCR LoD was used to estimate that of SENA by this (Figure 1 ) and employing few more commercial rRT-PCR diagnosis kits 171 in addition to BJ which was used in the titration experiment (Table S1 ). Totally 295 clinic samples 172 or specimens (mainly pharyngeal swabs) collected from 282 individuals were tested by rRT-PCR 173 followed by SENA detection (Table S3) . Except for asymptomatic carriers, all the cases of 174 uncertain analytic and false positive or negative readouts of rRT-PCR diagnosis were encountered 175 and finally confirmed or corrected by SENA detection. (Table S3) . SENA detection of these samples revealed not only the 12 suspected as negative 180 but also identified one more positive among the original 123 negative individuals, clearly a case 181 of false negative diagnosis (Table S3) . Besides, distinct rRT-PCR assay results, positive by HD 182 but negative by ZJ were shown for samples collected from 2 close contacts of COVID-19 patients 183 and apparently asymptomatic (ref to Table S3 ). However, the amplicons of both ZJ and HD were 184 shown as negative via SENA detection. All these ambiguous rRT-PCR amplicons (17 samples, ref 185 to Table S3 ) were finally analyzed by NGS, and the results were consistent with the SENA. 186 Noticeably, the rRT-PCR false-negative COVID-19 patient was symptomatically mild at the point (Table S3) , obtaining consistent results with those of SENA. 7 Consistently, the three SENA-negative individuals were finally excluded from SARS-CoV-2 201 infection after being rechecked by rRT-PCR after 24 hours (Table S3) . Based on above data, it is 202 clear, SARS-CoV-2 infection suspects with either rRT-PCR Ct values falling in the "grey zone" 203 or with clear patient-contact epidemiological history but negative rRT-PCR tests, are strongly 204 recommended to perform SENA detection to minimize the possibility of misdiagnosis. On the 205 other hand, in case an rRT-PCR-positive suspect does not demonstrate any COVID-19 clinic 206 symptoms and/or signs, SENA detection is also strongly recommended to eliminate either false-207 positive diagnosis or misdiagnosis of the so-called "asymptomatic carrier" or "asymptomatic 208 patient".

Besides of preventing false-negative or false-positive diagnosis, the highly sensitive property 210 of SENA may also assist in providing evidence of viral clearance for COVID-19 recovering 211 patients. A female patient in Dongfang Hospital (DF, Shanghai, China) was confirmed as COVID-212 19 positive by both rRT-PCR and CT scanning and showed ground-glass opacities mixed with 213 consolidation along the subpleural area (Figure 3) . Accordingly, the SENA test was positive with 214 the mixFCratio of 1.43. After the hospitalization, the patient was further analyzed by rRT-PCR at 215 two time points, obtaining all negative results with bilateralnasal and pharyngeal swab specimens.

However, the mixFCratios of SENA for some of her specimens were 1.64, 1.36 and 1.00, 217 respectively, indicating that the virus was contained and yet to be cleared. On the sixth day, both 218 rRT-PCR and the corresponding SENA detection for all of her specimens were negative and these 219 results were confirmed by NGS and consistent with her normal CT scanning results (Figure 3) . 220 Thus, she was discharged from the hospital and safely back to home. Similar cases were found in 221 Jinan of Shandong Province, China, where the fecal samples from two recovering COVID-19 222 patients were tested negative by rRT-PCR but clearly positive by SENA (Table S3) . Considering 223 a certain percentage of the recovered patients discharged from hospitals were reported to be re-224 detectable positive (RP) (22), the incomplete clearance of the SARS-CoV-2 virus ahead of 225 discharge might be one of the possible causes. Therefore, it could be necessary to consider more 226 sensitive detection approaches such as SENA as a potential index of viral clearance.

To reconfirm and/or improve the cut-off values for SENA mixFCratio, the ambiguous Ct 228 values were re-estimated using the regression functions derived from the rRT-PCR assays with 229 titrated standard RNA templates (Figures S4 and S5) , and then the Ct values (both estimated and 230 detected) were plotted against the corresponding mixFCratios (Figure 4) CoV-2 infection. In addition, the cut-off value for SENA mixFCratio remains unchanged as 1.145 236 for positive diagnosis while slightly increased to 1.068 for negative (Figure 4) , which is supposed 237 to further increase along with the clinic applications.

Instead of developing a closed CRISPR-Dx system, which, ideally, should be comprised of 239 both target nucleic acids amplification and CRISPR-Cas-based trans-cleavage assays, SENA was 240 created here to match the commercially available and widely applied rRT-PCR kits, and to solve 241 the uncertainty challenge of the rRT-PCR "grey zone" in COVID-19 diagnosis. Although there 242 are dozens of commercial rRT-PCR kits using distinct PCR primers and probes, their 243 corresponding SENA detection kits can be easily designed and prepared following the instructions 244 provided in this study. Considering the fact that qPCR is the most popular MDx system and SENA 245 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted June 5, 2020. Data and materials availability: 269 "All data is available in the main text or the supplementary materials." 270 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

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be indicated by their atypical clinic symptoms or signs. For all these cases, the corresponding rRT-347 PCR products can be sent to another physically isolated room for SENA analysis and the ambiguity 348 may be clarified by SENA with its positive and negative cut-off mixFCratio. The real-life data 349 related to these scenarios revealed in this study are shown in the figure and details are illustrated 350 in the text. RJ, JNCDC and SZII are the names of the hospitals and the number indicates the overall 351 number of patients identified. While P140 was a patient in DF hospital, and two distinct samples 352 from P140 were identified to be false-negative. For details, please ref to Supplementary Table S3. 353 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

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The copyright holder for this preprint this version posted June 5, 2020. in the text. Notice that the SENA negative cut-off was set as mix-FCratio=1.020 in this figure on 358 the basis of the titration assay of the standard RNA templates but was adjusted to 1.068 along with 359 the increase of the clinic applications (Figures 1 and 4, Supplementary Table S3 ).

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The copyright holder for this preprint this version posted June 5, 2020. . Table S3 . Other clinic data indicated that P140 365 is a COVID-19 patient with mild clinic symptoms. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

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The copyright holder for this preprint this version posted June 5, 2020. Tables S1 to S3 412 413 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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Reactions were conducted in a 25-μl reaction mixture following the instructions offered by the commercial 425 suppliers of the reaction kits (ref to Supplementary Table S1 ). Usually, the reaction cycle parameters were set 426

as reverse transcription at 50 °C for 10 min, denaturation at 95 °C for 5 min, then followed by 45 cycles of 427 amplification, i.e., 95 °C for 10 s and 55 °C for 40 s. 428 429 2. SENA reagents and detection. 430

Candidate crRNA guide sequences with the "TTN" PAM sequences were designed as shown in Supplementary Table  432 S1, and the crRNAs were prepared following the procedures previously described (11) . Except the target DNA, the 433 2× SENA reagent comprises of 2× NEB buffer 3.1, 500 nM LbCas12a (Tolo Biotech.), 1 μM synthesized crRNAs 434

(for each specified target and thus varies according to the RT-qPCR kit supplier, Table S1 ), 1 μM FQ-reporter, and 1 435 U/μl RNase inhibitor (TaKaRa). 436

To avoid the aerosol contamination of the MDx laboratory, after the RT-qPCR reaction, their products must be 438 transferred to a physically isolated room to perform SENA detection. It is also important to choose proper SENA 439

detection reagents corresponding to the RT-qPCR kits. To prepare a 20-μl SENA reaction system, with corresponding 440 positive and negative controls, 2-μl PCR products and 8-μl RNase-free H2O were mixed with 10-μl 2× SENA reagent, is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 5, 2020. . https://doi.org/10.1101/2020.06.02.20119735 doi: medRxiv preprint 20 55-μl extracts per sample. After RT-qPCR assays employing 5 μl of the extracts from each sample, all of the samples 468 were shown to be SARS-CoV-2 negative. The remaining 50-μl extracts of each sample were mixed together and 5 μl 469 of the mixture was once again analyzed by RT-qPCR and confirmed to be SARS-CoV-2 negative. Then, the mixed 470 nucleic acid extract was used as the dilution buffer (totally about 2 ml) for serial dilution of the SARS-CoV-2 RNA 471 standard stocks, generating desired concentrations (i.e., 5, 2, 1, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05, 0.025, 0.01, 0.005 472 copies/μl), and 5 μl of each of the diluted solutions were used as templates for RT-qPCR analysis, forming gradient 473 template concentrations (i.e., 25, 10, 5, 4, 3, 2, 1, 0.5, 0.25, 0.125, 0.05, 0.025 copies/Rx). 474

We analyzed 9 replicas for each of the concentrations of 1 and 0.5 RNA template copies/Rx while 6 replicas for each 476 of the rest concentrations (Supplementary Figure S1) . 477

RT-qPCR reaction kit was supplied by BJ (Table S1) Three readout parameters were compared as shown in Figure S1 (original data in Table S2 ). The slope (increase of 488 fluorescence/min) represents the reaction rate of Cas12a trans-cleavage activity, but in this experiment, it represents 489 neither the initial rate of the enzyme under limited substrate condition nor the pure first-order reaction rate varies 490

according to the substrate concentration, particularly, demonstrated in cases of high template concentration, in which, 491 the slope goes down along with the increase of the template concentration. In addition, when the substrate 492 concentration is low, the slope of SENA is hard to be distinguished from that of the negative control, ending with 493 ambiguous cut-offs. The FC (the fold of change of fluorescence between that of the sample over that of the negative 494 control at certain time point, usually 5-30 min) does show clear differences between the positive amplicons from that 495 of negative control, and it also shows certain quantitation character particularly at the low concentration templates 496

cases. Because of these properties, FC has been a parameter used by a few users of CRISPR-Dx (24). However, it 497 seems that the absolute value of the FC usually varies along the reaction time and sometimes it is influenced by the 498 change of the fluorescence signal of the negative control. Besides, it is difficult to determine the "best choice" of the 499 FC recorded at certain time points, which may cause confusing in clinic applications. It is clear, we need a stable 500 readout which reflects the dynamic process and the quantitative correlation of SENA reaction with the low 501 concentration of the templates on one hand and should be robust and accurate for clinical diagnosis on the other hand. 502

We defined FCratio, which is the ratio between FC's of SENA detection at 10 min versus 5 min after the beginning 503 of the fluorescence reading. It not only measures the fluorescence change of SENA against the negative control 504 background so that the quantity of the amplicons, particularly at the low concentration range may be represented, but 505 also normalizes the slope of the fluorescence change of SENA to eliminate the complex background differences. As 506 shown in Figure S2 , FCratio significantly amplified the positive signals and represents the quantitation of the 507 amplicons at low template concentrations to certain extent. 508

As shown in Figure S1 , the capacity of the three SENA reactions are compared. It is obvious that N-SENA is the 509 least sensitive one, while although O-SENA seems much more sensitive than that of N-SENA and largely comparable 510

to that of mix-SENA, its signal at the very low template concentration range seems uncertain in some cases. Therefore, 511

for clinic application, mix-SENA is the choice. 512

The quality of the Ct values vs the concentration of the standard templates for RT-qPCR of the systematic titration 514 experiment were analyzed both empirically and statistically. Firstly, as shown in Table S2 and Figure S1 , the 515 amplification efficiencies of the two genes represented by the valuable Ct readouts were different. The apparently 516 lower sensitivity of the N gene amplification is in contrast with the clinic experiences and might due to the difference 517 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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in the property of the templates used in different experiments (laboratory standard RNA template vs clinical real viral 518 template). Secondly, although linear regression can be readily made between the Ct-values and the log10 (conc) 519

( Figure S3A ) as that of the previously published tests, the quality of the regression as judged by the R 2 s (Figure S3A ) 520 and the residues ( Figure S3B ) are clearly suboptimal likely due to the limited number of replicas in the 521 experimentation. On the other hand, this titration was designed with taking at least the two most fundamental limitation 522 factors about the sensitivity of COVID-19 RT-qPCR diagnosis into consideration, i.e., the sampling ambiguity and 523 the influence of the biological/chemical contaminants from the clinic samples. Therefore, the data will be used for 524 determination of the LoD for RT-qPCR and SENA (Materials and Methods 4.4 and Figure S2 ), and the regression 525 function will be used to estimate the "apparent Ct" values of the samples with Ct-values greater than 45 (no 526 amplification signal) but their SENA detection is positive (Materials and Methods 4.5 and Figure S4) . 527

The LoD values for RT-qPCR (N-Ct and O-Ct) and SENA (N-FCratio, O-FCratio and mix-FCratio) were estimated 529 based on the systematic titration employing standard RNA templates ( Figure S1 and Table S2 ). The fractions of 530 positive replicates versus the number of target molecules (copies) per reaction for N and O gene of COVID-19 were 531 plotted and used the sigmoidal function (1) to fit the data via R (software version 3.5.0). The 95% confidence intervals 532

were derived by bootstrapping the model residues and were visualized by R (software version 3.5.0) with built-in 533 ggplot2 library (21). 534

employing the data from the systematic titration 536

Since PCR product increased exponentially with the initial concentration of the sample (x), and the Ct value of RT-538 qPCR parameter (y) was inversely correlated with the initial concentration, especially in the range of low copy number 539 (low template concentration) samples, the power function equation (a<1) should be suitable for the data fitting. 540

However, some of the experimental groups included very low initial sample concentrations (<1 copy/Rx), those 541 amplification efficiencies should be different (particularly affected by sampling ambiguity) from that of the groups 542 with high initial template concentration. Therefore, the power function formula with four parameters (Y=aX n +bX m ) 543

was used to match all the experimental group data to obtain a more accurate data model (Functions 1, Figure S4 ). 544

The exponential function (first order association kinetics of the interaction between a substrate and an enzyme, 546

Y=a+b(1-e cX )) is used to fit the data of FCratio against the concentration of the templates. At low concentration 547

(especially when the concentration is less than 2 copies/Rx), the FCratio is positively correlated with the template 548

concentrations. However, when the template concentration reaches to 2 copies/Rx and more, the FCratio does not 549 increase accordingly and the curve tend to be flatted out. In addition, as FCratio have been already normalized by the 550 fluorescence signal of the negative background, it is stable and, in this case, we give the parameter Y0 being set as a 551

constant value between 0.9 to 1 (Functions 2, Figure S5 ). 552

In practice, quite significant portions of the clinic positive samples detected by mix-SENA with their FCratio readings 554

higher than the positive cut-off, but with a negative PCR Ct value (>40-45, depending on the scenario). Under certain 555 circumstances, people may be interested to learn the copy number of the templates for the corresponding RT-qPCR 556 assays or even the "probable" Ct values of these assays. With the aid of the above-mentioned two regression functions 557

(1 in Figure S4 and 2 in Figure S5 ), these data could be estimated. One may firstly substitute the Y in Function 2 by 558 the measured FCratio value and the corresponding X can be calculated representing the "estimated concentration of 559 the template). Then this X value can be used to estimate the corresponding "estimated Ct-value" as the Y of Function 560

1. We estimated all the ambiguous Ct-values of the positive amplicons and plotted them against their corresponding 561 mix-FCratio (Figure 4) . All the real and estimated Ct values for both N and O genes are plotted against the 562

corresponding FCratio values of mix-SENA as X axis. An exponential decay function (with X0 = 1; When X≤X0, 563

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Ct=∞; otherwise, one phase decay) fits well to all the data (R 2 =0.9238) and is used for analyzing the clinic data and 564

adjust the cut-off values accordingly (Figure 4) . 565 566

The detection of anti-SARS-CoV2 antibodies was executed by the point-of-care microfluidic platform integrating a 568 home-made fluorescence detection analyzer (Suxin, Shanghai, China). A total of 10-μl plasma was added into the 569 loading chamber of microchip followed by the addition of 70-μl sample dilution buffer. After incubation for 15 min 570 at room temperature, the microchips were loaded onto the fluorescence detection analyzer, and fluorescence signal 571 was detected from the analyzer, following the manufacturer's instruction. 572 573 574 575

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The copyright holder for this preprint this version posted June 5, 2020. (Table S2) . . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 5, 2020. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 5, 2020. . https://doi.org/10.1101/2020.06.02.20119735 doi: medRxiv preprint Note: N/A, Detection of this gene that was not supported by the kit. "+", Positive; "-", negative; /, PCR product was not analyzed by NGS. Samples in aquamarine background stood for SENA negative but rRT-PCR positive or uncertain diagnosis. Samples in red background stood for SENA positive but rRT-PCR negative or uncertain diagnosis.

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CRISPR-Cas12a 37 target binding unleashes indiscriminate single-stranded DNase activity

Programmed DNA destruction by miniature CRISPR-Cas14 enzymes

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