key: cord-103422-ys846i99
authors: Xu, Xinhui; Luo, Tao; Gao, Jinliang; Lin, Na; Li, Weiwei; Xia, Xinyi; Wang, Jinke
title: CRISPR-Assisted DNA Detection, a novel dCas9-based DNA detection technique
date: 2020-05-13
journal: bioRxiv
DOI: 10.1101/2020.05.13.093062
sha: 
doc_id: 103422
cord_uid: ys846i99

Nucleic acid detection techniques are always critical to diagnosis, especially in the background of the present COVID-19 pandemic. The simple and rapid detection techniques with high sensitivity and specificity are always urgently needed. However, the current nucleic acid detection techniques are still limited the traditional amplification and hybridization. To overcome the limitation, we here develop a CRISPR/Cas9-assisted DNA detection (CADD). In this detection, DNA sample is incubated with a pair of capture sgRNAs (sgRNAa and sgRNAb) specific to a target DNA, dCas9, a signal readout-related probe, and an oligo-coated solid support beads or microplate at room temperature for 15 min. During this incubation, the dCas9-sgRNA-DNA complex is formed and captured on solid support by the capture sequence of sgRNAa and the signal readout-related probe is captured by the capture sequence of sgRNAb. Finally the detection result is reported by a fluorescent or colorimetric signal readout. This detection was verified by detecting DNA of bacteria, cancer cell and virus. Especially, by designing a set of sgRNAs specific to 15 high-risk human papillomaviruses (HPVs), the HPV infection in 64 clinical cervical samples were successfully detected by the method. All detections can be finished in 30 minutes at room temperature. This detection holds promise for rapid on-the-spot detection or point-of-care testing (POCT).

with collateral cleavage activity of single-stranded DNA (ssDNA) was used to develop DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter) (28, 29) and HOLMES (a one-HOur Low-cost Multipurpose highly Efficient System) (30, 31) . Immediately, the HOLMESv2 (HOLMES2.0) and CDetection technology were developed by using the collateral cleavage activity to single-stranded DNA (ssDNA) of Cas12b (also known as C2c1) (32, 33) . These methods are demonstrated to have attomolar sensitivity and single-base specificity. The ultrahigh sensitivity of these methods partially rely on the signal amplification resulted from collateral cleavage activity of related Cas enzymes. These methods are therefore used to detect various pathogenic viruses such as Zika virus (ZIKV) (24, 25) , dengue virus (DENV) (24) , human papillomavirus (HPV) (28) , Japanese encephalitis virus (JEV) (32) , African swine fever virus s (ASFV) (34) , and Mycobacterium tuberculosis (MTB) (35) . Recently, these techniques are striving to detect SARS-CoV-2 (36) (37) (38) (39) (40) (41) . However, these CRISPR Cas-based detection methods typically require DNA pre-amplification with polymerase chain reaction (PCR) (31) , reverse transcription-RPA (RT-RPA) (25) , recombinase polymerase amplification (RPA) (25, 27, 28, 34, 35) , Loop-mediated isothermal amplification (LAMP) (32, 39) , reverse transcription-LAMP (RT-LAMP) (32, 39) , and asymmetric PCR (32) . These pre-amplifications also partially contribute to the ultrahigh sensitivity of these methods.

Additionally, SHERLOCK also needs reverse transcription and T7 in vitro transcription when detecting RNA (27) . Such amplification dependence still makes these methods difficult to become simple, rapid and portable diagnostic tools.

In this study, we develop a new DNA detection method based on CRISPR/Cas9 named as CRISPR/Cas9-assisted DNA detection (CADD). In this method, a pair of capture sgRNAs (sgRNAa and sgRNAb) are designed for a target DNA. SgRNAa and sgRNAb harbor a different 3′ terminal capture sequence. When target DNA is bound by a pair of dCas9-sgRNA complexes, the dCas9-sgRNA-DNA complex will be captured on surface of beads or microplate via annealing between an oligonucleotide coupled on solid supports and capture sequence of sgRNAa. Then the captured dCas9-sgRNA-DNA complex is reported by a kind of signal reporter captured by the capture sequence of sgRNAb. This method was validated by detecting DNA of bacteria, cancer cell and virus. Especially, by designed a set of sgRNAs specific to 15 high-risk human papillomaviruses (HPVs), this study successfully detected 64 clinical cervical samples with this method. Of importance, a target DNA can be rapidly detected in less than 30 minutes using a signal readout of fluorescent hybridization chain reaction (HCR). This method has its unique advantages over the current methods such as free of enzyme, pre-amplification, modular heaters, simplicity and rapidness.

SgRNA was designed with online sgRNA design software Chop-Chop (http://chopchop.cbu.uib.no/), using hg19 as the reference genome. The designed sgRNAs are shown in Table S1 . Each DNA target had a pair of sgRNA, named sgRNAa and sgRNAb, respectively. According to the designed sgRNA, primers (Table   S2 ) were synthesized to amplify sgRNA template by PCR using our previous protocol (42) . The PCR-amplified sgRNA template has a T7 promoter sequence. SgRNA was then prepared by in vitro transcription using sgRNA template as previously described (42) . The prepared sgRNA had a 5′-end 20 bp target DNA-specific sequence and a 3′-end capture sequence. SgRNAa had a 3′-end capture sequence named as Capture 1 that was used to anneal with the capture oligonucleotide immobilized on the surface of the magnetic beads or microplate. SgRNAb had a 3′-end capture sequence named as Capture 2 that was used to anneal with a signal reporter-associated oligonucleotide. 5 

The full-length L1 fragments of 15 high-risk HPV (hrHPV) were cloned in the pMD plasmid (Takara) to  prepare HPV plasmids, including: pMD-HPV16, pMD-HPV18, pMD-HPV31, pMD-HPV33,   pMD-HPV35, pMD-HPV39, pMD-HPV45, pMD-HPV51, pMD-HPV52, pMD-HPV56, pMD-HPV58,  pMD-HPV59, pMD-HPV66, pMD-HPV68 , and pMD-HPV73.

Oligonucleotides FAM-hairpin-1 and FAM-hairpin-2 (Table S3) (Table S3 ) or sgRNA. The reaction was kept at RT for 20 min and detected by agarose gel electrophoresis.

A biotin-modified oligo RE-flanking (Table S3) 

All procedures used in this research were performed according to the Declaration of Helsinki. This study was approved by the Ethics Committee of Jinling Hospital (Nanjing, China). All participants were recruited from the Jinling Hospital with informed consent. The clinical HPV detection was performed by Jinling Hospital (Nanjing, China) using a human papillomavirus genotyping (type 23) detection kit (PCR-reverse dot hybridization method) (Asia Energy Biotechnology, Shenzhen). The gDNA extraction and HPV detection (a PCR-reverse dot hybridization method) were all performed with this kit. DNA was first tested by hospital and then the left DNA was brought to our laboratory. Two batches of clinical DNA samples were detected by CADD.

The Beads-HCR detection reaction (20 µL) M FAM-hairpin-2 and 1× Binding buffer) and incubated at RT for 15 min in rotation. The beads were then dropped on slide glass and covered with a cover glass. The beads were imaged with a fluorescence microscope. The beads images were analyzed with Image pro. 6 Single-target Beads-ELISA detection reaction (20 µL) contained 1× Binding buffer, 15 nM sgRNAa, 15 nM sgRNAb, 30 nM dCas9 protein, 1-4×10 4 Beads@oligo, 0.5 µM oligo re-biotin (Table S3) Beyotime). The beads were incubated at RT for 10 min. The microplate was read at 630 nm and imaged with a BioRad gel imager in staining-free mode.

An amino-modified oligo RE-NH 2 ( ; where N is the number of targets detected. The reaction was added to the oligo-coated microplate.

The microplate was incubated at RT for 15 min on a horizontal mixer and then washed three times with the washing buffer. The microplate was added with 100 µL of washing buffer containing 8 ng of HRP-conjugated Streptavidin and incubated at RT for 3 min. The microplate was then washed three times with the washing buffer. The microplate was then added with 30 μ L of washing buffer and 50 μ L TMP chromogenic solution for ELISA (Beyotime). The microplate was incubated for 10 min. The microplate was read at 630 nm and imaged with a BioRad gel imager in staining-free mode.

To explore the feasibility of CADD, we first designed a Beads-HCR method (Fig. 1A) , in which the fluorescent hybrid chain reaction (HCR) was used as signal readout. A pair of sgRNA were designed for a target DNA. Different from the traditional sgRNA, the sgRNA used by CADD was designed to have a short extended 3′ terminal capture sequence that can anneal with other functional oligonucleotides. In detection, a pair of dCas9-sgRNA (dCas9-sgRNAa and dCas9-sgRNAb) first binds the target DNA. The dCas9-sgRNA-DNA complex is then captured onto the surface of beads via annealing between the capture sequence on the sgRNAa and a complementary oligonucleotide coupled on beads. The beads are then washed on magnet and then two HCR Hairpins (Hairpin 1 and Hairpin 2) are added. The capture sequence of sgRNAb can anneal with Hairpin 1 to initiate HCR. Because the Hairpins are labeled by fluorescein, fluorescent signal can be produced on the surface of beads by HCR.

To investigate whether the designed HCR reaction is feasible, we tested the prepared Hairpin 1 and Hairpin 2 in liquid-phase HCRs with sgRNAa plus sgRNAb, sgRNAa, sgRNAb, and initiator oligo, 7 respectively. The results show that HCR reaction was only initiated by sgRNAb (Fig. S1 ), indicating that the capture sequence of sgRNAb annealed with Hairpin 1. The oligo Initiator (Table S3 ) is a positive control that can also anneal with Hairpin 1 to initiate HCR (Fig. S1 ).

With the reliable Hairpins and sgRNAb, we first detected the HPV16 DNA (pMD-HPV16) with Beads-HCR CADD using sgRNAs targeting HPV16 (sgRNA16). The results show that pMD-HPV16 can be quantitatively detected by the method (Figs. 1B and 1C) . To investigate the specificity of Beads-HCR detection, we then detected pMD-HPV16 and pMD-HPV18 with Beads-HCR CADD using sgRNA16, sgRNA18, and sgRNAct, respectively. SgRNAct is an equimolar mixture of sgRNAs of 15 hrHPVs. The results show that the two genotypes of HPVs can be specifically by the method (Figs. 1D and 1E ).

To further explore the feasibility and specificity of In order to investigate whether Beads-HCR CADD can be used to detect HPV DNA in human gDNA, we next detected gDNAs of three cervical cancer cell lines using sgRNA16, sgRNA18 and sgRNAct. HeLa and SiHa cells are known with HPV18 and HPV16 infection, respectively, and C-33a is known without HPV infection. The results indicate that the HeLa gDNA was detected by sgRNA18 and sgRNAct, the SiHa gDNA was detected by sgRNA16 and sgRNAct, and C-33a gDNA was not detected by any sgRNA (Fig. S2 ). These results indicate that Beads-HCR CADD is qualified for detection of more complicated DNA sample than HPV plasmid. In order to investigate whether Beads-HCR CADD can be used to detect clinical sample, we then detected 31 clinical DNA samples with sgRNAsp of 15 hrHPVs and sgRNAct. The results are shown as Fig. 4 and supplementary File 2. We compared the HPV detection results of these 31 clinical samples tested by Beads-HCR CADD and PCR-reverse dot hybridization method that was performed by Jinling Hospital (Fig. 4C ). In comparison with the hospital tests, the hrHPV infection (yes or no) and genotype are accurately detected by Beads-HCR CADD with 100% sensitivity and specificity. Importantly, the Beads-HCR CADD also found multiple infections in samples 2, 11, and 37 that were not detected by the PCR-reverse dot hybridization (Fig. 4C ). These multiple infections were confirmed by a PCR re-detection (PCR-rd) (Fig. S3 ), in which HPV45 and HPV59 infection were detected by PCR using primers specific to the two HPVs (Supplementary method and Table S3 ). These results indicate that Beads-HCR CADD can be used to detect hrHPV infections in clinical samples with high sensitivity and specificity. Finally, to investigate whether DNA other than virus DNA could be detected by Beads-HCR CADD, we also detected two types of other DNA. One is bacterium DNA and the other is human oncogenic DNA (Supplementary methods). The sgRNAs targeting T7 RNA polymerase DNA and oncogenic telomerase reverse transcriptase (TERT) promoter were designed (Table S1 ). The results indicate that the T7 RNA polymerase DNA fragment could be quantitatively detected by Beads-HCR ( Fig. S4A and S4B ). In addition, the subsequent detection of gDNA from two different E. coli (DH5α and BL21) indicate that the T7 RNA polymerase DNA in BL21 gDNA could be also specifically and quantitatively detected by Beads-HCR CADD ( Fig. S4C and S4D ). The DH5α gDNA that contains no T7 RNA polymerase DNA did not produce fluorescence signal even at the highest amount ( Fig. S4C and S4D ). The detection of TERT 8 promoter DNA indicate that the ontogenetic TERT promoter can be specifically detected by Beads-HCR CADD using a sgRNAs targeting mutated TERT promoter (Fig. S5) . The mutant TERT promoter causes expression of telomerase, which results in malignant cell proliferation in more than 90% of cancers. It should be noted that the detected TERT promoter has only one base difference between the wild-type and mutant genotype (Fig. S6) , indicating that CADD has high specificity that can discriminate single nucleotide polymorphisms (SNP).

Because Beads-HCR CADD is dependent on fluorescent microscope, we then expect to realize a CADD with visual readout. We therefore designed a Beads-ELISA form of CADD (Fig. 5A) . In this format of CADD, after dCas9-sgRNA binds target DNA and the dCas9-sgRNA-DNA complex is captured on beads surface via sgRNAa, a biotinylated oligonucleotide is captured by sgRNAb. The HRP-labeled streptavidin is then associated with biotin. Finally a soluble chromogenic substrate TMB is used to develop color signal.

The detection results can thus be read either qualitatively with naked eyes or quantitatively with microplate reader.

As a pilot assay, we first detected pMD-HPV16 with Beads-ELISA CADD. The results indicate that pMD-HPV16 can be quantitatively detected by this method (Fig. 5B-5D ). In this assay, pMD was also used as a negative control. It cannot produce color even at the highest concentration (100 pM). We then detected 15 hrHPVs with this method using various sgRNAsp and sgRNAct. The results indicate that each hrHPV were specifically detected by its cognate sgRNAsp and sgRNAct ( Fig. 6A and 6B ). The negative control pMD was not detected by any sgRNA. To check if this method can detect multiple infections, we subsequently detected seven mixtures of two different hrHPVs using various sgRNAsp and sgRNAct. The results reveal that the simulated multiple infections were specifically detected by this method (Figs.6C and   6D ). To further check the detection specificity, we finally detected several hrHPVs and clinical DNA samples with this method. The results demonstrate that each hrHPV was detected by its cognate sgRNAsp and sgRNAct (Fig. 6E) ; however, all single or mixed clinical DNA samples were not detected by any sgRNA (Fig. 6E) . Because the clinical DNA samples were selected from the Beads-HCR CADD-detected samples, we focused on investigating if false positive can be produced by sgRNA when detecting clinical samples. The results reveal that all selected clinical samples were not detected by any sgRNA, further indicating the high specificity of this method.

To further simplify the Beads-ELISA CADD and also try a new solid support other than beads, we designed a Microplate-ELISA form of CADD (Fig.7A) . In this method, a capture oligonucleotide is coupled in microplate. The dCas9-sgRNA-DNA complex will be captured in microplate through annealing between the 3'-end capture sequence of sgRNAa and the capture oligonucleotide. The signal reporting system is completely the same as the Beads-ELISA CADD.

To verify this method, we first detected pMD-HPV16 using sgRNA16. The results indicate that pMD-HPV16 can be quantitatively detected by this method (Figs. 7B and 7C) . The negative control pMD did not generate a signal at the highest concentration (Figs. 7B and 7C). We then detected 33 new clinical samples with this method using various sgRNAsp and sgRNAct. The results reveal that all samples were detected by this method (Fig. 8A and 8B ). In comparison with the results obtained by hospital tests using the PCR-reverse dot hybridization, the Microplate-ELISA CADD accurately detected the infections of 15 hrHPVs (yes or no) and genotypes with 100% sensitivity and specificity. Additionally, the Microplate-ELISA CADD also identified more multiple infections in Samples 2, 5, 15, and 33. To confirm 9 the test results, we re-detected the five samples with Microplate-ELISA. The results confirm that the Microplate-ELISA CADD can more accurately identify multiple infections in clinical samples than the current method used in clinics ( Fig. 8D and 8E ).

In this study, we designed a new CRISPR/Cas9-based DNA detection technique and validated it by detecting three types of DNA including bacteria DNA, human cancer cell DNA and virus DNA. We also verified three forms of CADD method by detecting 15 hrHPV plasmids and as many 64 clinical cervical samples. These investigations demonstrate the feasibility and reliability of CADD method. In comparison, CADD has its unique advantages over the current CRISPR-based nucleic acid detection methods. Different from the current widely known CRISPR-based nucleic acid detection methods that mainly rely on Cas enzymes with collateral cleavage activity such as Cas13a, Cas12a, Cas12b, and Cas14, we develops a new DNA detection method based on a most widely used Cas protein, CRSIPR/Cas9, that has no collateral cleavage activity. CADD relies on neither the specific enzymatic activity nor non-specific collateral cleavage activity of CRISPR/Cas proteins as all the current CRISPR-based methods. Therefore, CADD is mechanistically distinct from the current CRISPR-based methods (such as SHERLOCK, DECTECTR, and HOLMES). CADD employs dCas9 that has no enzymatic activity. CADD uses the dCas9-sgRNA as a DNA-binding complex with high sequence specificity.

This study indicates that CADD can be used to detect and type various target DNA molecules with high simplicity, sensitivity and specificity. Although the current CRISPR-based methods are reported to have ultrahigh detection sensitivity (aM), they are all challenged by tedious pre-treatments to DNA/RNA samples including various amplifications using RPA, LAMP, PCR, asymmetric PCR and in vitro transcription. These pre-amplifications not only complicate the detection process and increase the detection time and cost, but also may increase false negative, because pre-amplification with various DNA polymerases may introduce mutations into DNA that is then enzymatically detected by various Cas proteins. Importantly, amplicon spread and contamination is always a serious issue in all detection spots. CADD needs no any pre-treatment to DNA sample. Beads-HCR CADD is an enzyme-free method.

Beads/Microplate-ELISA CADD only need a widely used enzyme HRP to develop signal. All three forms of CADD methods can be performed without depending on any modular heaters. Another advantage of CADD is free of traditional hybridization at high temperature. The whole CADD process is carried out at room temperature. The time-consuming and instrument-dependent hybridization and amplification are key limitations to the on-the-spot application of the current nucleic acid tests. Due to these advantages, CADD will have wide applications in future DNA detection.

The CADD signal readout can be versatile. In this study, two forms of CADD signal readouts were verified, fluorescent and colorimetric readout. The Beads-HCR CADD uses fluorescent readout by employing the fluorescently labeled HCR hairpins. The Beads/Microplate-ELISA uses colorimetric readout, in which the TMB color development catalyzed by streptavidin-coupled HRP is used as a visual readout. The Beads/Microplate-ELISA CADD is an instrument-free test. In addition, the Microplate-ELISA assay allows automatic measurement of hundreds of samples on standard plate reader in a high-throughput format. The whole detection process of Beads-HCR and Microplate-ELISA can be finished in 30 minutes, holding promise for rapid on-the-spot detection or point-of-care testing (POCT). In fact, other forms of CADD signal readouts can be realized by a few changes of the solid support and signal reporter, such as lateral flow readout, nano-gold colorimetric assay, and molecular beacon-HCR. 10 The high specificity of CADD has close relationship with the high sequence specificity of dCas9-sgRNA as a DNA-binding complex. Additionally, the high specificity of CADD is also dependent on its unique detection mechanism, in which a pair of sgRNAs commonly determine the final detection results, providing a double-insurance sequence-specific detection. This overcomes the potential false positive results resulted from potential off-target binding of one dCas9-sgRNA. This problem still challenges all current CRISPR-based methods including SHERLOCK, DECTECTR and HOLMES, in which only one-site target cutting can activate a collateral cleavage activity. Due to the signal amplification produced by collateral cleavage activity, detections based on such one-site activation may be prone to false positives.

HPV is a double-stranded DNA virus that is closely related to the pathogenesis of cervical cancer, anal cancer and other cancers (43) . There are about 100 different types of HPV. According to different carcinogenic capabilities, HPV is divided into high-risk HPV (hrHPV) and low-risk HPV (lrHPV). The most common hrHPVs in the world are HPV16 and HPV18, which cause about 70% of cervical cancers (44, 45 

We Fig. S1 .

Evaluation of HCR reaction. Fig. S2 . Detection of gDNA of cervical cancer cell lines using Beads-HCR CADD. Fig. S3 . Detection of HPV45 in samples 2 and 37 and HPV59 in sample 11 using specific PCR amplification. Fig. S4 . Detection of T7 RNA polymerase DNA using Beads-HCR CADD. Fig. S5 .

Detection of TERT promoter DNA using Beads-HCR CADD. s g R N A c t s g R N A 1 6 s g R N A 1 8 s g R N A 3 1 s g R N A 3 3 s g R N A 3 5 s g R N A 3 9 s g R N A 4 5 s g R N A 5 1 s g R N A 5 2 s g R N A 5 6 s g R N A 5 8 s g R N A 5 9 s g R N A 6 6 s g R N A 6 8 s g R N A 7 3

pMD-HPV16 pMD-HPV18 pMD-HPV31 pMD-HPV33 pMD-HPV35 pMD-HPV39 pMD-HPV45 pMD-HPV51 pMD-HPV52 pMD-HPV56 pMD-HPV58 pMD-HPV59 pMD-HPV66 pMD-HPV68 pMD-HPV73 s g R N A c t s g R N A 5 1 s g R N A 1 6 s g R N A 1 8 s g R N A 3 1 s g R N A 3 3 s g R N A 3 5 s g R N A 3 9 s g R N A 4 5 pMD pMD-HPV16+pMD-HPV18 pMD-HPV18+pMD-HPV31 pMD-HPV31+pMD-HPV33 pMD-HPV33+pMD-HPV35 pMD-HPV35+pMD-HPV39 pMD-HPV39+pMD-HPV45 pMD-HPV45+pMD-HPV51 

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