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CRISPR/Cas9 triggered isothermal amplification for site-specific nucleic acid detection Mengqi Huang, Xiaoming Zhou, Huiying Wang, and Da Xing Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04542 • Publication Date (Web): 20 Dec 2017 Downloaded from http://pubs.acs.org on December 20, 2017

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Analytical Chemistry

CRISPR/Cas9 triggered isothermal amplification for site-specific nucleic acid detection Mengqi Huang, Xiaoming Zhou*, Huiying Wang, and Da Xing* MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, PR China * To whom correspondence should be addressed. Tel: (+86-20) 8521-1436; Email: [email protected]; Correspondence may also be addressed to Xiaoming Zhou. Tel: (+86-20) 8521-1436; Email: [email protected]. ABSTRACT: A novel CRISPR/Cas9 triggered isothermal exponential amplification reaction (CAS-EXPAR) strategy based on CRISPR/Cas9 cleavage and nicking endonuclease (NEase) mediated nucleic acids amplification was developed for rapid and site-specific nucleic acid detection. CAS-EXPAR was primed by target DNA fragment that produced by cleavage of CRISPR/Cas9, and the amplification reaction performed cyclically to generate a large number of DNA replicates which were detected using a real-time fluoresces monitoring method. This strategy that combines the advantages of CRISPR/Cas9 and exponential amplification showed high specificity as well as rapid amplification kinetics. Unlike conventional nucleic acids amplification reactions, CAS-EXPAR does not require exogenous primers, which often cause target-independent amplification. Instead, primers are firstly generated by Cas9/sgRNA directed site-specific cleavage of target, and accumulated during the reaction. It was demonstrated this strategy gave a detection limit of 0.82 amol and showed excellent specificity in discriminating single-base mismatch. Moreover, the applicability of this method to detect DNA methylation and L. monocytogenes total RNA was also verified. Therefore, CAS-EXPAR may provide a new paradigm for efficient nucleic acid amplification and hold the potential for molecular diagnostic applications.

Rapid and sensitive detection of nucleic acids with high specificity is significant for biological research and clinical diagnosis.1,2 As a routine technique for nucleic acids detection, polymerase chain reaction (PCR) has been widely used in basic research due to its excellent reliability and sensitivity.3 However, the requirement for a thermal cycling instrument make it difficult to be applied in low-resource settings. Recently, various isothermal amplification methods, such as nucleic acid sequence-based amplification (NASBA),4 rolling circle amplification (RCA),5-7 strand displacement 8,9 amplification (SDA), loop-mediated isothermal amplification (LAMP),10 recombinase polymerase amplification (RPA)11 and exponential amplification reaction (EXPAR),12 are becoming increasingly important for nucleic acid analysis.

amplification methods for rapid and sensitive long DNA or RNA detection, which introduce exogenous probes (such as three-way junction probe or beacon probe) to generate triggers, take the risk of target-independent amplification.20,21 Other strategies for long nucleic acid detection hold well fidelity, but they were limited to detect target sequences that contain a nicking-enzyme recognition site.22,23 The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems as an RNA-mediated adaptive immune system which can protect organisms from invading viruses and plasmids were first found in the bacteria and archaeal.24 The type II CRISPR/Cas9 system which merely need the Cas9 endonuclease and an engineered single guide RNA (sgRNA) can induce cleavage of double-stranded (ds-) DNA with a protospacer adjacent motif (PAM, a short DNA sequence of the form 5’-NGG-3’, where ‘‘N’’ = any nucleotide).25 Cas9 from S. pyogenes has been proved to be an extremely efficient mean of DNA targeting for various applications such as genome editing,26-28 genetic screening,29 gene expression modulation30,31 and imaging.32 Moreover, recent in vitro work has demonstrated that single-strand (ss-) DNA and ssRNA can also be cleaved by Cas9/sgRNA when a separate PAM-presenting oligonucleotide (PAMmer) that is antisense to the ssDNA or ssRNA was present.33,34 Therefore, Cas9 can serve as a programmable nucleic acid ‘nicking’ tool for site-specific cleavage of target ssDNA sequence. Taking advantage of unique strengths of Cas9/sgRNA, the characteristic sequence within long nucleic

As a promising nucleic acid amplification method, EXPAR which involves polymerase directed strand extension and nicking endonuclease (NEase) induced single strand nicking has been successfully applied in the analysis of nucleic acids and proteins.13-15 Compared with other isothermal amplification methods, EXPAR exhibits distinct advantages of higher amplification efficiency and rapid amplification kinetics.12,16 Up to now, EXPAR method has widely used for microRNA (miRNA) detection in which the short miRNA is served as primer to initiate amplification.17-19 However, EXPAR is rarely used for long DNA or RNA detection, because the characteristic sequence within long DNA or RNA with hundreds or even thousands of nucleotides cannot directly act as a primer to initiate EXPAR. Current exponential

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Recombinant RNase inhibitor and RNase-free water were purchased from Takara. SYBR Green I was purchased from Life Technologies (Carlsbad, CA). The real-time fluorescence detection was performed using CFX 96 real-time PCR detection system (Bio-Rad). NanoDrop 2000 UV-Vis spectrophotometer. Production of sgRNA. The DNA template of sgRNA was prepared by fill-in PCR which was performed with two long primers: a 65-nt oligonucleotide containing the T7 promoter and an 80-nt oligonucleotide ecoding the 3’ tail sequence of sgRNA, between which has 20-nt length complementary. The PCR product was firstly purified by SanPrep Column PCR Product Purification Kit and then used as template for T7 RNA polymerase-mediated transcription reaction. The transcription reaction of sgRNA was performed with purified DNA template for 6 h at 37 °C. T7 transcription product, namely, sgRNA was purified by RNAclean Kit (Tiangen). The sgRNA was measured by NanoDrop 2000 for detailing its concentration and subsequently stored at -80 °C. In vitro Cas9 cleavage assay. The cleavage assay was carried out with 10 µL of reaction mixture containing 1×reaction buffer (20 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA), 50 nM Cas9 nuclease, 50 nM sgRNA, different concentrations of target ssDNA and 2 nM PAMmer. Firstly, Cas9 nuclease and sgRNA was pre-incubated in 1×reaction buffer in a total volume of 8 µL at 25 °C for 5 min. Then 1 µL target ssDNA and 1 µL PAMmer were added into the mixture. Finally, the mixture was incubated at 37 °C for 30 min and the reaction was quenched by heating to 95 °C for 5 min. The cleavage reaction products will be subsequently used for exponential amplification

Scheme 1. Schematic for the principle of the CAS-EXPAR. Firstly, Cas9/sgRNA complex mediates site-specific cleavage of ssDNA substrates producing cleaved fragments (X). Secondly, the X hybridizes with the EXPAR template, and is extended by DNA polymerase from its 3’ terminus along the template. Then, the formed duplex is nicked by NEase, and a copy of X is released from the template by Vent (exo-) DNA polymerase. The dissociated X will trigger a new amplification circuit. acid could be selectively broken and the produced fragment can be used to other analytical purposes.

EXPAR protocol. Before EXPAR, the reaction solution was prepared separately as part A and part B. The part A solution contained Nt.BstNBI nicking enzyme, Vent (exo-) DNA polymerase, Thermopol buffer and SYBR Green I. The part B solution contained amplification template, dNTP mix, NEBuffer 3.1 and Cas9 cleavage reaction products. After preparation, part A and part B were mixed immediately and added RNase-free water to 10 µL which contains 250 µM dNTP mix, 0.4 U/µL Nt.BstNBI, 0.05 U/µL Vent(exo-) DNA polymerase, 2 ×SYBR Green I, 1 ×Thermopol buffer (20 mM Tris-HCl, pH 8.8, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1 %Triton (X-10)), 0.5 ×NEBuffer 3.1 (25 mM Tris-HCl, pH 7.9, 50 mM NaCl, 5 mM MgCl2 and 0.5 mM dithiothreitol) and 0.1 µM EXPAR template. The mixture was incubated at 55°C in real-time PCR detection system and fluorescence intensity was monitored at 1 min intervals.

Here, we developed a CRISPR/Cas9 triggered exponential amplification method (CAS-EXPAR) which combines the advantages of site-specific cleavage of Cas9/sgRNA and rapid amplification kinetics of EXPAR. This method does not require exogenous primers, which may cause target-independent triggering. Instead, the ‘primers’ are firstly generated by site-specific cleavage of target DNA sequence and accumulated during the reaction. By incorporating a real-time fluorescence intensity analysis method, sensitive DNA detection with CAS-EXPAR could be realized within 1h. Moreover, by coupling with bisulfite conversion that converts single-base methylations into single-base mutations, this strategy could also realize site-specific DNA methylation detection. Therefore, CAS-EXPAR can serve as a versatile nucleic acid detection strategy for molecular diagnostic applications.

RNA extraction and reverse transcription. Total RNA was isolated from L. monocytogenes bacterial cells with the RNAiso Plus reagent according to the manufacturer’s instructions, and the obtained total RNA was assessed by gel electrophoresis and measured by NanoDrop 2000 UV-Vis spectrophotometer. The reverse transcription (RT) reactions was carried out with 20 µL of reaction mixture containing 1 µM RT primer, 1×RT buffer, 0.25 mM each of dNTPs, 10 U/µL M-MLV reverse transcriptase, 1 U/µL RNase inhibitor and extracted total RNA. The RT reaction was incubated at 42 °C for 60 min. Then 5 U RNase H and 1×RNase H reaction buffer was added in the mixture and incubated at 37 °C for 30 min.

EXPERIMENTAL SECTION Reagents and instruments. All oligonucleotides were synthesized and purified by Sangon Biological Engineering and Technology & Services Co., Ltd. (Shanghai, China). The sequences of probes were listed in Table S1. Vent (exo-) DNA polymerase, Nt.BstNBI, Cas9 Nuclease, T7 RNA polymerase, M-MLV reverse transcriptase, RNase H, ribonucleotide (NTP) solution mix, and deoxynucleotide (dNTP) solution mix were all purchased from New England Biolabs (Beijing, China).

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Analytical Chemistry and DNA polymerase can sequentially extends the cleaved dsDNA, meanwhile displaces the X. The released X can hybridize with another T to trigger amplification again. Through the circuit reaction of CRISPR/Cas9 mediated cleavages, NEase cleavage, polymerase extension and strand displacement, a large amount of dsDNA could be synthesized. Therefore, the CAS-EXPAR can report the presence of the target through real-time fluorescent tracking. Feasibility analysis. At first, the recognizing and cleaving ability of the designed Cas9/sgRNA complex was verified by polyacrylamide gel electrophoresis (PAGE) and silver staining analysis. A 637bp hlyA gene fragment was used as the substrate. After dsDNA substrate was incubated with Cas9/sgRNA complex at 37°C for 0.5 h, two clear band of cleavage with predicted length(one is 400bp, the other is 237bp) could be seen (Figure S1). The result showed that designed sgRNA is able to active the cleavage ability of Cas9. Subsequently, a 41 nt ssDNA target was used to test the feasibility of CAS-EXPAR using real-time fluorescence intensity analysis method. The cleaved site was designed at dinucleotide junction between position 3 and 4 of ssDNA-sgRNA hybrid. As shown in Figure 1A, it was confirmed that, in the present of 10 fmol target ssDNA, the fluorescence intensity increased fastly and reached a plateau in 10 min, which displayed a typical sigmoidal response. Control experiments were performed in CAS-EXPAR system in a lack of Cas9 nuclease or target ssDNA. The point of inflection (POI: the time corresponding to the maximum slope in the fluorescence intensity curve), which was defined in analogy to threshold cycle in real-time PCR analysis, was used for quantitative detection of the target. Comparatively, both of the controls without Cas9 enzyme or target show delayed increase of fluorescence intensity (POI > 20 min). These results show that the exponential amplification reaction could be triggered by Cas9/sgRNA mediated site-specific cleavage of target. Additionally, the proposed strategy was also verified using non-denaturing polyacrylamide gel electrophoresis (PAGE) (Figure 1B). Compared with negative controls without target or Cas9, the experiment sample displayed a new band after Cas9/sgRNA cleavage and taking exponential amplification at 55 °C for 20 min. The new band could be the short oligonucleotide X conducted by CAS-EXPAR, therefore, confirming the feasibility of proposed CAS-EXPAR strategy.

Figure 1. Analysis of isothermal exponential amplification triggered by Cas9/sgRNA cleavage. (A) Real-time fluorescence intensity curves of CAS-EXPAR. Target ssDNA (10 fmol) was used for triggering exponential amplification in the present (Cleaved) or absent (Uncleaved) of Cas9. Blank sample was negative control without target ssDNA. (B) Non-denaturing PAGE analysis of CAS-EXPAR products. Bisulfite conversion. 1.3 µg methylated or nonmethylated DNA was subjected to bisulfite conversion with EpiMark bisulfite kit (NEB) according to manufacturer’s instructions. The bisulfite converted DNA was measured by NanoDrop 2000 for detailing its concentration and then used in CAS-EXPAR. Statistical analysis: Statistical analyses were performed with Origin Lab version 8. Each experiment was repeated at 3 times for each samples tested. The results were present as mean ±standard deviation, unless otherwise indicated.

RESULTS AND DISCUSSION General assay scheme of CAS-EXPAR. The principle of developed CAS-EXPAR technology for nucleic acids detection is illustrated in Scheme 1. A L. monocytogenes hemolysin (hly) gene fragment with a “NCC” region that complement to PAM sequence is used as the target model. Firstly, a sgRNA is designed to contain three segments: a 20-nt guide sequence, a 30-nt repeat: anti-repeat duplexes region, and three tracrRNA stem loops. The guide sequence that is an essential part for target recognition is complementary to the 3’-downstream sequence of the “NCC” region of target DNA, the 30-nt repeat: anti-repeat duplexes region and three tracrRNA stem loops are necessary to trigger the Cas9 cleavage activity. The sgRNA can bind with Cas9 to form an activated Cas9/sgRNA complex which can carry out site-specific cleavage of target. Secondly, an EXPAR template (T) is designed to perform the subsequent exponential amplification circuits, which possesses a central NEase recognition sequence and two same flanking regions (X’) that complementary to the Cas9 cleaved target fragment (X). Through these design, the X hybridizes with X’ at the 3’-end region of T to form a complex (TX) and serves as a primer for DNA polymerase to synthesize a duplex with a complete ds-NEase recognition region and two X sequences. Then, NEase nicks the complementary strand of T in formed dsDNA,

In CAS-EXPAR, PAMmer is an important element for amplification efficiency. On the one hand, PAMmer can bind with target and acts as an allosteric regulator to significantly increase the cleavage efficiency of Cas9/sgRNA,34-36 thus facilitating exponential amplification. On the other hand, PAMmer compete with EXPAR template to bind with cleaved fragment X, thereby hinder the efficiency of exponential amplification. The process of strand hybridization and displacement in CAS-EXPAR were shown in Figure 2A. The rate constant k1 denotes the hybridization rate of PAMmer to target ssDNA, while k2 denotes the dissociation rate of PAMmer-ssDNA hybrid (PD). The kT and kP denote the rate constants of forward and reverse reaction rates of the strand displacement process, respectively. The ratio of the rate constants is given by kP/kT=e ∆G/KT

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Figure 2. Amplification efficiency analysis of CAS-EXPAR. (A) The process of strand hybridization and displacement in CAS-EXPAR. The displacement of PAMmer by EXPAR template is mediated by toehold region of PX. (B) Real-time fluorescence intensity curves on analyzing 10 fmol target ssDNA and negative controls in the presence of various length of PAMmer. (C) Histogram of ∆POI values with various length of PAMmer. Error bars are the standard deviation of triplicate measurements.

Figure 3. Sensitivity analysis of CAS-EXPAR. (A) Real-time fluorescence intensity curve with various concentration of target ssDNA (10 fmol, 1fmol, 100amol, 10amol and 1amol). Blank sample is negative control without target. (B) Correlation of POI values to logarithmic concentrations of target ssDNA. Error bars are the standard deviation of triplicate measurements.

Where ∆G is the free-energy change in duplex formation for the toehold region. Previous researches have shown that ∆G is mainly influenced by length of toehold domain: the longer toehold region is, the smaller ∆G becomes, consequently, the smaller the rate of kP/kT becomes.37,38 Therefore, longer toehold region, namely shorter length of PAMmer contribute to obtain high kP/kT value for fast strand exchange. However, if the length of PAMmer is too short, it cannot bind with target DNA stably, thereby; the cleavage efficiency will be greatly reduced, leading low amplification efficiency. Here, we use three variant length of PAMmer to investigate the influence of PAMmer on CAS-EXPAR: 11-nt PAMmer (PAMmer 11), 9-nt PAMmer (PAMmer 9) and 7-nt PAMmer 7 (PAMmer 7), therefore, the corresponding length of toehold region was 8-nt, 10-nt and 12-nt respectively. Real-time fluorescence intensity analysis of target ssDNA and negative controls with different length of PAMmer was shown in Figure 2B. The ∆POI values corresponding to different length of PAMmer were shown in figure 2C. The ∆POI value was the difference of POI values between positive sample and negative control in the presence of same PAMmer. As shown in figure 2C, PAMmer 9 and PAMmer 11 showed almost same ∆POI values, indicating a

comparable reaction rate. Although PAMmer 7 was more favorable for toehold-mediate strand exchange than PAMmer 9 and PAMmer 11, the unstable hybridization between PAMmer 7 and target DNA led to a weak cleavage of Cas9/sgRNA, thereby, resulting in lower ∆POI value. As previous report showed that there was still weak cleavage of ssDNA by Cas9/gRNA in the absent of PAMmer,34 we challenged to detect ssDNA without PAMmer and the result showed a slightly lower ∆POI value compared with PAMmer 11. This result indicated that CAS-EXPAR can not only be applied in ssDNA detection with PAMmer, but also be used for ssDNA detection with slight sacrifice of efficiency in the absent of PAMmer. Sensitivity of CAS-EXPAR. Subsequently, we tested the sensitivity of CAS-EXPAR by analysing different concentrations of target ssDNA using real-time fluorescence monitoring system. As shown in Figure 3, the fluorescence intensity curves all displayed the sigmoidal shape and the POI values were linearly corresponding to the logarithm (log) of target ssDNA concentration in the range from 1 amol ~ 10 fmol. The correlation equation was obtained as POI=

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Figure 4. Single-base mismatch analysis with CAS-EXPAR. (A) Detail sequence of four single base mismatched targets. (B) Real-time fluorescence intensity curves of full-matched target and four single-base mismatched targets with sgRNA 20. (C) Histogram of ∆POI values of 10 fmol target ssDNA, MM 1 and MM 3 in the presence of sgRNA 20 and three truncated sgRNA (sgRNA 18, sgRNA 17, sgRNA 16). (D) Discrimination factor of CAS-EXPAR with four sgRNAs on MM1 and MM3. Error bars are the standard deviation of triplicate measurements.

-32.76-2.87 lg Atarget (mol) with a correlation coefficient of 0.979, where Atarget is the amount of target ssDNA. The detection limit was estimated to be as low as about 0.82 amol, which is comparable or superior to many reported methods for detecting characteristic sequence within genome DNA.39-42 Although CAS-EXPAR may be less sensitive than some isothermal amplifications, it can avoid primers induced non-specific triggering. Compared with standard EXPAR, it is simpler for CAS-EXPAR to detect specific site within long DNA, but it is hard for standard EXPAR. A table of comparison between our method and traditional isothermal DNA amplification methods was shown in Table S2.

the target DNA at the 3th nucleotide of ssDNA-sgRNA hybrid would not only disturb cleavage of Cas9/sgRNA, but also produce a 3’ flap when cleaved fragment hybridized with EXPAR template which inhibited DNA polymerase extension, thereby, resulting in low amplification efficiency. To investigate specificity of CAS-EXPAR, we designed four DNA sequence (MM1, MM2, MM3 and MM4) with a single-base mutation at different site (Figure 4A). Among them, MM3 was designed to have a single-base mutation at cleavage site that is the 3th nucleotide of ssDNA-sgRNA hybrid. It was observed that MM1, MM2 and MM4 showed almost the same fluorescence increase as full-matched target with 20-nt guide sequence of sgRNA (sgRNA 20), while MM3 displayed delayed fluorescence increase compared with full-matched target (Figure 4B). The results indicated that CAS-EXPAR can discriminate base changes at cleavage site. Previous researches showed that Cas9-mediated DNA cleavage was governed by conformation shift of Cas9 structure, which was stabilized by interactions between the guide RNA and the protospacer at or near the 14th–17th nucleotide positions from the PAM.43

Specificity of CAS-EXPAR. High specificity of CAS-EXPAR was attributed to site-specific cleavage of Cas9/sgRNA and sequence-dependent DNA polymerase extension. When Cas9/sgRNA hybridized with target DNA, it would cleave the phosphodiester bond between 3th and 4th nucleotide of ssDNA-sgRNA, producing fragments with a hydroxyl at its 3’-end. Subsequently, the fragment hybridized with EXPAR template and was extended along EXPAR template from its 3’ end by DNA polymerase. Mismatches in

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DNA methylation that is a covalent base modification have strong correlation with various aspect of gene control and disease 46,47. Currently, bisulfite conversion as chemical methods for methylation recognition was widely used in DNA methylation analysis 48,49. Treatment of DNA with sodium bisulfite converts nonmethylated cytosine into uracil while leaving 5-methylcytosine unchanged, which transformed the difference of methylation into difference of single-base mismatch 50,51. Due to high specificity of CAS-EXPAR, we hypothesized that CAS-EXPAR can detect DNA methylation by coupling with bisulfite conversion. The principle of DNA site-specific methylation detection of CAS-EXPAR was shown in Figure 6A. After treatment of bisulfite, all of cytosine on ssDNA were converted into uracil except the 5-methylcytosine, which appeared single-base mutation between nonmethylated and methylated DNA. The 5-methylcytosine was designed at cleavage site, and the guide sequence of sgRNA and EXPAR template was designed according to bisulfite converted methylated ssDNA sequence.

Figure 5. Real-time fluorescence detection of L. monocytogenes mRNA with CAS-EXPAR. 2.5 µg total RNA were reversely transcripted to cDNA followed by CAS-EXPAR in the presence or absent (Uncleaved) of Cas9. Blank sample were negative control without total RNA. Due to Cas9 cleavage specificity can be significantly improved using truncated sgRNA,44,45 we speculate that base changes at cleavage site or 14th–17th nucleotide positions from the PAM would make bigger influence on CAS-EXPAR with truncated sgRNA. Hence, full-matched target, MM1 (single-base mismatch at 14th nucleotide positions from the PAM) and MM3 were chosen to investigate the specificity of CAS-EXPAR with four length (20-nt, 18-nt, 17-nt and 16-nt) of guide sequence of sgRNA (sgRNA 20, sgRNA 18, sgRNA 17 and sgRNA 16). The results were shown in Figure 4C. Compared with sgRNA 20, all of these truncated sgRNA showed decreased ∆POI values for full-matched target, MM 1 and MM 3. The result suggested that the cleavage efficiency was decreased with decline of guide sequence of sgRNA. The single-base mismatch discrimination factor (DF, defined as the ratio of ∆POI values between perfect matched target and single-base mismatched DNA) was used to evaluate the discrimination ability of Cas9/sgRNA for single-base mutations. It was observed that both of MM1 and MM3 showed increased DF values with Cas9/sgRNA 16 (Figure 4D), which meant that Cas9/sgRNA 16 was more sensitive on single-base mutations. Besides, Cas9/sgRNA 16 showed high DF values (6.5) on MM3, indicating that Cas9/sgRNA 16 can clearly discriminate base mutation at cleavage site. Therefore, the proposed CAS-EXPAR strategy showed high specificity which can discriminate single-base mismatch.

Figure 6. DNA methylation detection with CAS-EXPAR. (A) Process of methylation DNA detection with CAS-EXPAR. When nonmethylated and methylated ssDNA were treated with bisulfite, all of cytosine was converted into uracil except the 5-methylcytosine, resulting in single-base mutation between nonmethylated and methylated DNA. The single-base mutation can be discriminated by CAS-EXPAR through two processes: recognition of Cas9/sgRNA and DNA polymerase mediated primer extension. (B) Real-time fluorescence curves of methylated and nonmethylated DNA in the present of four sgRNAs (sgRNA 20, sgRNA 17 and sgRNA 16) with variant length of guide sequence.

CAS-EXPAR for detection of L. monocytogenes mRNA. To further verify the application feasibility of the proposed CAS-EXPAR strategy, we applied CAS-EXPAR to detect foodborne pathogen L. monocytogenes hly mRNA from extract samples (Figure 5). The mRNA was reversely transcripted to cDNA followed by CAS-EXPAR. Control experiments were performed in CAS-EXPAR system in a lack of Cas9 nuclease or total RNA. It was observed that POI values of 2.5 µg and 1.25 µg total RNA samples were meaningfully lower than negative controls, suggesting that CAS-EXPAR could successfully apply to L. monocytogenes mRNA detection. DNA methylation detection with CAS-EXPAR. Cytosine

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NOTES

When bisulfite treated nonmethylate DNA was used in CAS-EXPAR, there would be a U-G mismatch on Cas9/sgRNA recognition and DNA polymerase extension process. As shown in Figure 6B, there was obvious difference of POI values between methylated DNA and nonmethylated DNA with Cas9/sgRNA 20 and Cas9/sgRNA 17, indicating that the single cytosine methylation could be distinguished by CAS-EXPAR. Besides, sgRNA 17 showed bigger differences of POI values between methylated DNA and nonmethylated DNA than sgRNA 20, which indicated better discrimination ability for DNA methylation. Therefore, by coupling with bisulfite conversion, CAS-EXPAR could apply in site-specific DNA methylation detection.

The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China [21475048], the National Science Fund for Distinguished Young Scholars of Guangdong Province [2014A030306008], the Project of Guangzhou Science and Technology Plan [201508020003], and the Program of the Pearl River Young Talents of Science and Technology in Guangzhou [2013J2200021], the Special Support Program of Guangdong Province (2014TQ01R599), and the Outstanding Young Teacher Training Program of Guangdong Province (HS2015004)

CONCLUSIONS

REFERENCES

We present a novel nucleic acid detection method based on CRISPR/Cas9 mediated site-specific cleavage and isothermal exponential amplification. This method appears to have several promising features for research and diagnostic applications as follows: (1) the proposed strategy which combines the advantages of site-specific cleavage of Cas9/sgRNA and rapid amplification kinetics of EXPAR shows high specificity in discriminating single-nucleotide mismatch as well as high amplification efficiency. The high specificity of CAS-EXPAR is derived from two sides: the recognition and site-specific cleavage of ssDNA substrate by Cas9/sgRNA at right site and sequence-dependent DNA polymerase extension from the 3’-end of cleaved fragment. (2) Unlike conventional nucleic-acid amplification reactions, CAS-EXPAR does not require exogenous primers which often cause target-independent amplification. Instead, ‘primers’ are generated by site-specific cleavage of target with Cas9/sgRNA and accumulated during the reaction. (3) Due to the programmable cleavage mode of Cas9/sgRNA, it seems that CAS-EXPAR is able to detect any site of target sequence. With this developed technology, as low as 0.82 amol target can be detected within 1h. Meanwhile, the applicability of this method to detect DNA methylation and L. monocytogenes total RNA are also demonstrated. In words, CAS-EXPAR strategy is a versatile method for diverse nucleic acid detection such as DNA, RNA and methylated DNA, which shows high specific and rapid amplification kinetics and holds great potential for applications in bio-analysis and disease diagnostics.

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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. The oligonucleotide sequences of CAS-EXPAR and Non-denaturing PAGE and silver staining analysis of Cas9/sgRNA cleaved products are included in Supporting Information.

AUTHOR INFORMATION Corresponding Author *Da Xing, PhD, Professor E-mail address: [email protected] *Xiaoming Zhou, PhD, Professor E-mail address: [email protected] Tel: +86-20-85210089; Fax: +86-20-85216052

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