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G-triplex: A new type of CRISPR-Cas12a reporter enabling highly sensitive nucleic acid detection

Source:sciencedirectAuthor:Tao Li

Highlights

CRISPR Cas12a trans-cleaves G-triplex structures.

• G-triplex formed by TBA11 is a promising Cas12a-based biosensing reporter.

• TBA11 reporter enhances the detection sensitivity up to 20 folds over normal ssDNA reporter.

• The LOD of our assay achieved 0.1 aM for the amplified HPV16 plasmid.

• A specificity and sensitivity of 100% and 94.7% was achieved in the detection of HPV patient samples.

Abstract

CRISPR Cas12a (Cpf1) trans-cleaves ssDNA and this feature has been widely harnessed for nucleic acid detection. Herein, we introduce a new type of Cas12a reporter, G-triplex (G3), and a highly sensitive biosensor termed G-CRISPR. We proved that CRISPR Cas12a trans-cleaves G3 structures in about 10 min and G3 can serve as an excellent reporter based on the cleavage-induced high-order structure disruption. G3 reporter improves the analytical sensitivity up to 20 folds, enabling the detection of unamplified and amplified DNA as low as 50 pmol and 0.1 amol (one copy/reaction), respectively. G-CRISPR has been utilized for the analysis of 27 PCR-amplified patient samples with HPV infection risk based on both fluorescence and lateral flow assays, resulting in 100% concordance between the two. In comparison with the clinical results, it achieved overall specificity and sensitivity of 100% and 94.7%, respectively. These results suggest that G-CRISPR can serve as a rapid, sensitive, and reliable biosensor, and could further expand the CRISPR toolbox in biomedical diagnostics.

1. Introduction

CRISPR/Cas system serves as an adaptive immunity system in bacteria and archaea to protect them against invading phages, virus and plasmids (Barrangou et al., 2007; Doudna and Charpentier, 2014).

During the interplay between pathogens and their hosts, CRISPR Cas machinery cleaves specific DNA with the guide of CRISPR RNA (crRNA) (Koonin et al., 2017). This property has been harnessed by scientists to revolutionize gene editing with the emergence of many CRISPR Cas proteins such as Cas9, Cas12, Cas13, and Cas14 (Abudayyeh et al., 2016; Harrington et al., 2018; Jinek et al., 2012; Pan et al., 2019; Zetsche et al., 2015). Recently, a growing body of research has demonstrated that

certain CRISPR Cas proteins exhibit collateral cleavage on non-specific targets (termed trans-cleavage) after the target-specific cleavage (termed cis-cleavage) (Abudayyeh et al., 2016; Gootenberg et al. 2017, 2018bib_Gootenberg_et_al_2018bib_Gootenberg_et_al_2017; Li et al., 2018a). Leveraging this special characteristic, numerous CRISPR-based point-of-care (POC) platforms (e.g., SHERLOCK) have been developed for human genotyping and pathogen detection, with high specificity and sensitivity (Fozouni et al., 2020; Gootenberg et al. 2017, 2018bib_Gootenberg_et_al_2018bib_Gootenberg_et_al_2017; Xiong et al., 2020a).

Among the CRISPR proteins, CRISPR Cas12a (also known as Cpf1) (Zetsche et al., 2015) offers promiscuous endonuclease activities that trans-cleave non-target ssDNA (but not dsDNA and RNA) (Chen et al., 2018; Li et al., 2018a; Swarts and Jinek, 2019). Using a ssDNA as the reporter, Chen et al. established the ‘DETECTR’ platform that achieved attomole (aM) sensitivity for human papillomavirus (HPV) detection (Chen et al., 2018). Li et al. implemented a similar ssDNA-FQ reporter and developed a system named ‘HOLMES’, and detected pseudorabies virus (RNA) and Japanese encephalitis virus (RNA) (Li et al., 2018b). Its sensitivity is around 10 aM for the detection of amplified targets. Thereafter, many other strategies have been proposed to optimize the performance of Cas12a-based platforms, such as to diversify the target types (e.g., combine aptamer to detect protein, small molecules besides DNA/RNA) (Dai et al., 2019; Liang et al., 2019; Xiong et al., 2020b; Yuan et al., 2020), to simplify the operation protocol (e.g., automatic sample extraction, one-pot detection) (Ding et al., 2020; Joung et al., 2020; Ramachandran et al., 2020), and to avoid the use of reader (Broughton et al., 2020; Shao et al., 2019; Wang et al., 2020b). Another key goal of the optimization is to improve the detection sensitivity, which could reduce the assay time without sacrificing accuracy during early viral infection (Jin et al., 2020; van Dongen et al., 2020; Wang et al., 2020a).

For example, researchers have introduced two crRNAs (Ding et al., 2020) and replaced Mg2+ with Mn2+ to increase Cas12a activity (Ma et al., 2020), both of which realized detection sensitivity of a few copies of nucleic acids. However, to our knowledge, current Cas12-based detection systems commonly use ssDNA as the reporter, which represents a limitation to further improve the sensitivity.

Recently, we have reported that the activated CRISPR/Cas12a system trans-cleaves DNA G-quadruplexes (G4s) (Li et al., 2020). However, it requires a long time (up to several hours) to cleave G4 structures due to their high stability. G-triplex (G3), a DNA motif like G4, has three-stranded noncanonical secondary structure that can be formed by Guanine-rich DNA sequences through Hoogsteen-like hydrogen bonds (Cerofolini et al., 2014; Limongelli et al., 2013). G3 was initially recognized as an intermediate form of G4 with potentially important cellular functions (Koirala et al., 2012; Li et al., 2013), and further studies revealed that a few sequences could form stable G3 structures (Cerofolini et al., 2014; Koirala et al., 2012; Limongelli et al., 2013), though not as stable as G4s (Jiang et al., 2015). G3 shares multiple properties as G4 (e.g., sensitive to many stimuli such as metal ions, hemin, thioflavin T) (Demkovicova et al., 2017; Ida et al., 2019), thus it has emerged as a popular transducer of various types of biosensors (Wang et al., 2013; Zhao et al., 2019). For example, a G3 sequence, obtained by truncating four bases (GGGA) from a G4 sequence (Zhang et al., 2014), appeared to show strong affinity with methylene blue (MB) and could be stabilized by MB. This characteristic was utilized to develop an electrochemical biosensor for sensitive detection of cocaine (Zhao et al., 2019).

In this work, we showed that CRISPR Cas12a could trans-cleave G3s and developed a sensitive nucleic acid detection platform based on this finding. We chose two typical G3 sequences, TBA11 (a truncated form of Thrombin Binding Aptamer or TBA) (Cerofolini et al., 2014; Limongelli et al., 2013) and telomere-G3 (Koirala et al., 2012) that were reported to be not so stable (Zhao et al., 2019), and investigated the cleavage activities of Cas12a on them. The results demonstrate that CRISPR Cas12a trans-cleaves both G3 structures in a few minutes and the cleavage rate is much higher than that of G4. We verified this trans-cleavage activity

with fluorescence spectroscopy, circular dichroism (CD) spectroscopy, gel electrophoresis, and nuclear magnetic resonance (NMR) experiments. Since G3 responds to various stimuli and can be degraded rapidly by the Cas12a system, it could be a promising reporter for building fast biosensors based on its high-order structural change before and after the cleavage. Accordingly, the G-CRISPR platform was developed by integrating CRISPR-Cas12a with the TBA11-G3 reporter. Using G-CRISPR, we successfully distinguished SARS-CoV-2 and SARS-CoV N-gene, and HPV16 and HPV18 L1-gene plasmids. The system achieved detection sensitivity of 50 pM and 0.1 aM (single copy per reaction) for the unamplified and amplified HPV16 plasmids respectively, improved up to 20 times over the ssDNA-reporter based Cas12a system. In addition, we demonstrated the successful detection of HPV16 and HPV18 in 27 patient samples using both fluorescent- and lateral flow-based G-CRISPR assays. The results are highly consistent with the clinical reports, resulting in a specificity and sensitivity of 100% and 94.7%, respectively. Overall, G-CRISPR offers high sensitivity and accuracy for nucleic acid detection and holds great potential to further expand the CRISPR Cas12a toolbox for biosensing.

G-triplex:A new type of CRISPR-Cas12a reporter enabling highly sensitive nucleic acid detection

2. Materials

G-triplex (G3), G-quadruplex (G4), and crRNA sequences were obtained from Sangon Biotech company (Shanghai, China). The SARSCoV-2 N-gene, SARS-CoV N-gene, HPV16 L1-gene and HPV18 L1-gene were amplified via polymerase chain reaction (PCR) from pUC57-SARS-CoV-2-N, pUC57-SARS-CoV-N, pUC57-HPV16-L1 and pUC57-HPV18-L1 vector, respectively. PCR Master Mix was purchased from Yeasen company (ShangHai, China), and PCR products of the plasmids were purified by gel extraction kit (Axygen company, New York, American) before use. The primers and vectors used were synthesized by Tsingke company (Beijing, China). DNA and RNA sequences used were listed in Supporting Information (Table S1). DNA agarose and nucleic acid gel stain were purchased from Yeasen company (Shanghai, China). TEMED, (NH4)2S2O8, urea, 30% acrylamide/bis-acrylamide solution and 10 × TBE Buffer were purchased from Bio-Rad Laboratories (Hercules, CA). Lachnospiraceae bacterium ND2006 Cas12a (CRISPR LbCas12a) was purchased from Magigen Biotech company (Guangzhou, China). Lateral flow strips (Milenia HybriDetect 1) were obtained from TwistDx (Cambridge, United Kingdom).


Source file:

G-triplex: A new type of CRISPR-Cas12a reporter enabling highly sensitive

nucleic acid detection

Tao Li , Rui Hu , Jianbo Xia , Zhichen Xu, Dongjuan Chen , Jinou Xi , Bi-Feng Liu ,Jiang Zhu , Ying Li , Yunhuang Yang , Maili Liu