Precision Detection of Rifampin-resistant rpoB L378R Mutation in Mycobacterium tuberculosis with CRISPR-Cas12a

Precision Detection of Rifampin-resistant rpoB L378R Mutation in Mycobacterium tuberculosis with CRISPR-Cas12a | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Precision Detection of Rifampin-resistant rpoB L378R Mutation in Mycobacterium tuberculosis with CRISPR-Cas12a Yuma Yang, Li Yang, Yue Zhu, Shuming Zhang, Hao Ma, Sihan Zhang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7257131/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Rifampin is the most effective drug in the treatment of tuberculosis.However, certain strains of MTB have developed resistance to rifampin, leading to the need for alternative treatment options.The rpoB gene mutations play a central role in MTB resistance to the rifampin, so it is essential to identify these mutations and efficiently treat rifampin-resistant MTB strains.This study developed a novel CRISPR-Cas12a platform integrated with recombinase polymerase amplification (RPA) and fluorescence detection to specifically identify the rpoB _L378R mutation associated with Rifampin resistance in (MTB).We found that this detection system was highly specific and did not cross-react with created reference samples containing the genomes of MTB H37Rv, Mycobacterium smegmatis, Mycobacterium aureus , and Escherichia coli. The CRISPR-Cas12a-based platform developed in this study was simple, sensitive, and specific for detecting the Rifampin-resistant MTB strain with the rpoB _L378R mutation.This suggested that it has potential for clinical applications in identifying MTB rpoB _L378R mutation. CRISPR-Cas12a Mycobacterium tuberculosis nucleic acid detection rifampin resistance rpoB Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), remains a major global public health threat and a leading cause of death from an infectious agent(Sotgiu et al., 2015 ).According to the World Health Organization (WHO) Global Tuberculosis Report 2023, an estimated 10.6 million people fell ill with TB worldwide in 2022, and 1.3 million people died from the disease (Organization, 2024).While approximately 85% of drug-susceptible TB patients can be cured with a standard 6-month drug regimen, drug-resistant TB (DR-TB), particularly multidrug- or rifampicin-resistant TB (MDR/RR-TB) which affected an estimated 3.3% of new TB patients and 17% of previously treated patients in 2022, poses enormous challenges to TB control and treatment efforts(Organization, 2024) .The development of drug resistance is primarily driven by mutations in specific bacterial genes.Key mutations conferring resistance to first-line drugs include those in the rpoB gene (e.g.codons S450L, L430P, H445D/N/Y) for rifampicin (RIF) and in the katG gene (e.g., codon S315T) or promoter region for isoniazid (INH) (Ma et al., 2021 ; Torres Ortiz et al., 2021 ; Welekidan et al., 2021 ).Notably, the rpoB gene is the target for the widely used Xpert MTB/RIF assay, which detects RIF resistance by identifying mutations, primarily within the core RIF resistance-determining region (RRDR).Mutations like S450L and H445D/N/Y within the RRDR, as well as others like L378R and R871H outside this core region, are frequently associated with RIF resistance (Ma et al., 2021 ).To effectively combat TB, especially MDR-TB, there is a critical need for rapid, sensitive, and accurate diagnostic tools that can detect TB infection and drug resistance early, thereby simplifying diagnosis and reducing costs, particularly in resource-limited settings. Nucleic acid amplification tests (NAATs) have significantly advanced this goal.Their advantages in accuracy and speed compared to traditional smear microscopy have led the WHO to strongly recommend NAATs as the initial diagnostic test for all individuals with signs and symptoms of pulmonary TB, effectively replacing microscopy as the primary diagnostic standard in most settings(Organization, 2021 , 2024).Polymerase chain reaction (PCR) is a foundational and widely used NAAT technology due to its high accuracy and specificity (Cao et al., 2017 ; Radmard et al., 2019; Shragai et al., 2022 ).However, conventional PCR often requires sophisticated laboratory infrastructure, expensive equipment, and trained personnel.Other established NAATs include Xpert MTB/RIF (and Ultra), line probe assays (LPAs), and DNA sequencing (Cai et al., 2020 ; Habte et al., 2016 ).Despite their utility, factors like cost, instrument dependency, and operational complexity can limit their accessibility in economically underdeveloped regions.To overcome these limitations, isothermal amplification methods such as recombinase polymerase amplification (RPA)(Ding et al., 2021 ; Miao et al., 2019 ) and loop-mediated isothermal amplification (LAMP) (Vanhomwegen et al., 2021 ) have been developed.These techniques operate at constant temperatures, potentially reducing reliance on complex thermocycling equipment.While RPA and LAMP have shown promise for detecting pathogens like ASFV and HBV(He et al., 2019 ; Miao et al., 2019 ; Vanhomwegen et al., 2021 ), their application for TB detection faces challenges, including achieving the necessary sensitivity and specificity in complex clinical samples, potential primer-dimer formation, and the need for robust and standardized protocols suitable for point-of-care use (He et al., 2019 ). Nucleic acid detection can also be accomplished with CRISPR-Cas systems, in which many Cas proteins including Cas12a, Cas12b, Cas13a and Cas14 can recognize dsDNA, ssDNA or RNA sequence under the guidance of single guide (sg) RNA (Aquino-Jarquin, 2019; Bhattacharyya et al., 2018; Chen et al., 2018 ; Ding et al., 2020 ; Gootenberg et al., 2018 ).CRISPR-Cas nucleic acid detection technology has been successfully applied for examining many diseases including Hepatitis B Virus (HBV)(Ding et al., 2021 ), SARS-CoV-2(Broughton et al., 2020 ; Huang et al., 2021 ; Joung et al., 2020 ), HIV(Ding et al., 2020 ), Dengue and ZIKA(Ackerman et al., 2020 ; Kaminski et al., 2020 ; Myhrvold et al., 2018 ) ; in addition, CRISPR-Cas-based detection technologies have also achieved single nucleotide recognition(Kim et al., 2020 ; Liu et al., 2021 ; Myhrvold et al., 2018 ).Therefore, CRISPR-Cas12a in combination with recombinase polymerase amplification (RPA) can be utilized to establish simple, fast, highly sensitive and specific detection methods for detecting the rifampin-resistant MTB strains with mutations such as rpoB_L378R(Kellner et al., 2019 ; Mustafa & Makhawi, 2021 ). In this study we hypothesized that the MTB drug resistance mutant gene rpoB_L378R could be used as a detection target, and the CRISPR-Cas12a system could detect single base differences in the mutant gene sequence.Our aim of this investigation was to establish a rapid, convenient, economical, highly specific, highly sensitive and visual detection method for MTB drug resistance point mutation by combining RPA isothermal amplification technology, fluorescence detection and paper chromatography.The result of this study lay the groundwork for the future development of MTB drug resistance mutation detection reagents for clinical applications. Materials and Methods Materials Major reagents involved in this study included EnGen LbCas12a (Cpf1) (New England BioLabs, M0653T), plasmid mini kit I (OMEGA, D6943-02), Luria broth (LB) media, Ampicillin (100 mg/mL), E.coli DH5αcompetent cell, NEBuffer2.1, polyacrylamide gel electrophoresis Gel Fast Preparation Kit (EpiZyme, PG114), Gel Extraction Kit D2500 (OMEGA, D2500-02), TaKaRa MutanBEST Kit (Takara, R401), QuickCutTM Hind III (TAKARA, 1615), TwistAmp® Basic Kit (TwistDx, TABAS03KIT), Milenia HybriDtect1 (TwistDX, Milenia01), UltraPure DNase/RNase -Free Distilled Water (Thermo, 10977015) and RNase inhibitor (Takara, 2313A).Thermo Fisher Nanodrop 1000 Spectrophotom was used to quantify nucleic acid.Fluorescence signals were detected by PerkinElmer EnVision Multimode Plate Reader, and fluorescence imaging was gained by Bio-Rad GEL Doc XR. 1.1 Methods Target sequence and crRNA preparation We selected rpoB gene sequence (from 760794 to 761540, long 753 bp) (GenBank accession NC_000962) as a research object.The wild-type rpoB gene of MTB H37Rv was downloaded from National Center for Biotechnology Information, and subsequently it was synthesized and cloned into topo plasmid (topo- rpoB ) by Beijing Ruibiotech.Both ends of the rpoB gene were synthesized with restriction enzyme cutting sites for Hind III.To obtain rpoB _L378R (CTG>CGG) target sequence, the topo- rpoB plasmid was performed a point mutation in codon 378 with mutation primers ( rpoB L378R_mut_F and rpoB L378R_mut_R) and TaKaRa MutanBEST Kit’s introduction. The crRNA was designed to pair with rpoB L378R mutant sequence by CRISPOR website (http://crispor.tefor.net/crispor.py), and the principle of designing crRNA was described previously(Kellner et al.2019).All nucleic acid sequences that were mentioned above were synthesized by Beijing Ruibiotech. Design of RPA amplification assay and primers To improve LbCas12a detection efficiency and sensitivity, RPA was performed with TwistAmp® Basic Kit.Four RPA primer pairs were designed at both ends of a mutant site of the rpoB L378R target sequence using Primer Premier 5.0 and refered to the TwistAmp Assay Design Manual.RPA primers length was designed from 19 nt to 35 nt, and the producing amplicon size was under 150 bp.Then, RPA primers were confirmed further by NCBI Primer-BLAST based on the RPA primers design principles.Before RPA assay, we selected the best primer pair based on the ratio and specificity of amplification, and then RPA amplification was performed strictly according to TwistAmp ® Basic Kit’s introduction.The primer sequences were synthesized by Beijing Ruibiotech and listed in Table 1. LbCas12a cleavage assay LbCas12a cleavage assay was carried out based on a previous protocol (Chen et al.2018 ; Shi Y et al.2022 ).with slight modifications.Briefly, Cas12a cleavage reaction was implemented with 400 nM Cas12a, 400 nM rpoB L378R-targeted crRNA, 1U/μL murine RNase inhibitor, and 100 ng rpoB L378R or rpoB _wild type in 1 NEB buffer 2.1.This mixture was incubated at 37℃ for 60 min and then analyzed by 2% agarose gel electrophoresis. FAM-BHQ1 fluorescence reporter detection The ssDNA FAM-BHQ1 reporters contained a poly (T) oligonucleotide sequence linked to fluorophore and quencher.In the FAM-BHQ1 fluorescence reporter detection assay, LbCas12a firstly bonded crRNA to form a LbCas12a-crRNA complex, and LbCas12a was activated when crRNA paired with the target DNA sequence, triggering the trans-cleavage activity of LbCas12a to cleave a non-target ssDNA reporter.In this assay there were 400 nM LbCas12a, 400 nM crRNA, 1 U/μL murine RNase inhibitor, 2.4 μM ssDNA FAM-BHQ1 fluorescent reporter and 100 ng target DNA rpoB L378R or rpoB _wild type in 1 NEB buffer 2.1.The reaction mixtures were quickly transferred to a light-proof 96-well plate at Multimode Plate Reader preheated 37 ℃, and fluorescence signals were measured every 5 min (FAM: λ ex =492, λ em =517).In addition, we also observed fluorescence intensity under light including UV, LED blue and no excitation light.In optimization of fluorescence detection conditions, we tried to improve fluorescent detection condition including incubating time (every 10 min for a total of 60 min), concentration of RPA primers (0.24, 0.36, 0.48, 0.60 μM) and ssDNA FAM-BHQ1 (1.6, 3.2, 4.8, 6.4 μM), and proportion of crRNA and Cas12a (1:1, 1:2, 2:1 based on 400 nM) to maximize detection effect. Lateral flow readout of Cas12a detection In lateral flow detection assay, the reaction mixture contained 400 nM LbCas12a, 400 nM crRNA, 1 U/μL murine RNase inhibitor, 1 μM ssDNA FAM-biotin reporters, RPA reaction mix of rpoB L378R or rpoB _wild and 1×NEB buffer 2.1type.After incubation for 40 min, 100 μL of HybriDetect 1 assay buffer (from Milenia HybriDetect 1 kit) was added.Subsequently, lateral flow detection was performed by placing a HybriDetect 1 lateral flow strip into a reaction system.After 1-2 min the colored readout was developed. Preparation and analysis of the bacterial samples rpoB -negative MTB H37Rv strains were inactivated at 100℃ for 10 min, and the genome was extracted using a Bacterial DNA Kit and preserved at -80℃.1 nM of MTB H37Rv genomic DNA, and 1 nM of rpoB L378R fragments were mixed as rpoB -positive.Other controls included the genomic DNA of HEK293T cell, M.smegmatis , M.aureus and E . coli .Noticeably, 2 μL of genomic DNA was subjected to RPA reaction and then to CRISPR-Cas12a detection in a fluorescence signal and lateral flow strip assay. The study was approved by the Medical Ethics Review Committee of Ningxia Medical University, and the reference number is 2020-531. Statistical Analysis Representative data were the mean ± standard deviation (SD) from triplicates.Statistical analyses were performed using GraphPad Prism 8.0.2 using two-way ANOVA.Significance was considered at * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 ; ns indicated no significance. Results CRISPR-Cas12a can be leveraged to detect a single nucleic acid mutation.We aimed to establish a rapid, convenient, economical, highly specific, highly sensitive and visual method for detecting MTB drug resistance point mutation.Thereby, we hypothesized that the CRISPR-Cas12a system could be used to detect MTB drug resistance mutant genes including rpoB L378R. dsDNA cleavage of ropB L378R based on CRISPR-Cas12a To accomplish the CRISPR-Cas12a cleavage detection on dsDNA rpoB L378R, as shown in Figure 1a, LbCas12a and crRNA were mixed to form a LbCas12a-crRNA complex at 37°C, the cis - and trans -acting cleavage activity of LbCas12a that could achieve targeted and no-targeted cleavage in turn was activated when crRNA and rpoB L378R bases paired perfectly (Chen et al.2018).Here, rpoB- L378 was mutated to L378R (CTG>CGG) (Figure 1b).To test the cis -cleavage activity of LbCas12a for rpoB L378R compared to rpoB _wild type, mutant specific crRNA was designed to distinguish rpoB L378R from rpoB _wild type (Figure 1c). Cis -acting cleavage lets a rpoB L378R sequence (753 bp) divide into two sections shown by 2 % agarose gel electrophoresis, resulting in a shorter band only shown in lane 4 (Figure 1c).This shorter DNA fragment represented a positive result from the cleaved rpoB L378R in the LbCas12a cleavage assay. Detection of rpoB L378R with CRISPR-Cas12a is simple, rapid and specific To visually identify rpoB L378R, LbCas12a trans-cleavage activity (collateral activity) was subjected to a non-targeted cleavage ssDNA FAM-BHQ1 fluorescence reporter assay (Figure 2a).In this assay, the RPA primers were first optimized.As shown in Figure 2b, there was a brighter and denser single band at about 150 bp in lane 6, indicating that this pair of RPA primers resulted in the best specificity and amplification efficiency among the tested pairs.Thereby, this pair of primers was used in the following experiments.We next verified LbCas12a trans-cleavage activity to a non-target DNA sequence (Figure 2a) and tested the specificity of point mutation discrimination by combining a ssDNA fluorescence reporter.As shown in Figure 2c, reaction 3 with rpoB L378R sequence, crRNA and LbCas12a produced a stronger fluorescence signal than that with rpoB _wild in reaction 7, and a fluorescence signal could be directly seen under blue LED or UV light.In addition, the specificity of the generated fluorescence signal was confirmed by a polyacrylamide gel electrophoresis (PAGE) assay, as shown in Figure 2c.A thick stripe with shorter DNA size was in lane 3 (Figure 2c), representing the cleaved ssDNA FAM-BHQ1 reporters with a strong fluorescence signal.In other reaction conditions, there were only weak bands with relatively longer DNA sizes in corresponding lanes, which might be attributed to the fluorescence quench of the intact uncut reporter.These results indicate that LbCas12a detection assay provides a simple, rapid and specific detection of rpoB L378R. To improve the CRISPR-Cas12a detection efficiency, we optimized fluorescence detection conditions.These included incubation time, the concentration of RPA primers and ssDNA FAM-BHQ1 fluorescence reporter, and the ratio of crRNA and LbCas12a.After identifying the mutants using the CRISPR-Cas12a fluorescence assay, we next tested if the incubation time of reaction could be reduced.The results (Figure 3a and b) showed that at the incubation time of 15-minutes (min) there was fluorescence under UV, LED blue light, and the strongest fluorescence was observed at the incubation time of 25 min.In optimizing concentrations of ssDNA FAM-BHQ1 reporters, the fluorescence could be observed under no excitation light in the reaction with 4.8 μM ssDNA FAM-BHQ1, and this concentration was used in the subsequent fluorescence assays (Figure 4a and b).In optimizing concentrations of RPA primers, we found that the primers at 0.24 μM gave the best result among the tested concentrations (Figure 5a and b).In particular, the fluorescence signal could be observed remarkably by naked eyes in Figure 5a.The ratio of crRNA to LbCas12a might play a critical role in improving cleavage activity and detection sensitivity.As shown in Figure 6a and b, at the ratios of 1:2 and 2:1 similar fluorescence signal emitted in real-time fluorescence curve, but we later observed that the ratio of 2:1 of crRNAs to LbCas12a gave a better result when crRNA was at 400 nM in the reaction system. Detection of ropB L378R with a CRISPR-Cas12a-original lateral flow detection assay To reduce instrument dependence and increase efficiency, we combined lateral flow strip with CRISPR-Cas12a.In the lateral flow assay, 5’-FAM and 3’-biotin-labeled 12-nt ssDNA reporter were combined with a lateral flow strip for fluorescence intensity readout (Figure 7a) (Chen et al., 2018; Moreno-Mateos et al., 2017)Similar to the fluorescence detection assay, with lateral flow strips we quickly detected rpoB L378R from rpoB _wild type with high sensitivity without requiring any signal detection instrument (Figure 7b).Band-intensity analysis showed a higher ratio of the test band to the control band resulting from the rpoB L378R samples than from the rpoB _wild (Figure 7b).These results together show that there are two methods of fluorescence signal detection and paper strip systems to detect rpoB L378R mutation with CRISPR-Cas12a, and these approaches are rapid, sensitive, and specific. Detection of rpoB L378R in biological samples using CRISPR-Cas12a Next, we evaluated the drug resistance mutation detection sensitivity and specificity with CRISPR-Cas12a-linked fluorescence detection and paper chromatography in different bacteria and human cells, which included HEK293T cell, M.smegmatis, M.aureus, E.coli, MTB H37Rv, and rifampicin-resistant MTB with rpoB L378R.Firstly, rpoB _wild and rpoB L378R fragments were mixed with M.smegmatis, M.aureus and E.coli genomes at molar concentration 1:1, respectively.The CRISPR-Cas12a assay showed high specificity for rpoB L378R (Figure 8a and b).Compared with the negative and wild-type control, the fluorescence intensity of rpoB L378R reaction system was the strongest, and the difference of paper chromatography was highly significant ( P <0.001).In detection systems of H 2 O, HEK293T, MTB H37Rv ( rpoB L378R), MTB H37Rv, M.smegmatis and M.aureus, RPA effectively amplified rpoB sequence with specificity and efficiency (Figure 8c).Fluorescence signal and paper chromatography detections based on CRISPR-Cas12a were used to identify MTB H37Rv ( rpoB L378R).As is shown in the fluorescence detection, the fluorescence intensity of the rpoB L378R reaction system was stronger than other reaction systems (Figure 8d).Moreover, the CRISPR-Cas12a system specifically recognized rpoB L378R with a sensitivity of 100 aM, and there was also a significant difference in the stripe gray assay ( P <0.01) (Figure 8e) Discussion In this report showed that CRISPR-Cas12a, together with RPA isothermal amplification can be utilized to detect the rifampin-resistant MTB strains with the mutations such as rpoB L378R.This approach we developed is rapid, convenient, economical, highly specific and sensitive for identifying rpoB L378R ; in addition, it did not need expensive equipment, and it is accurate because in this system the collateral activity of Cas12a enables highly specific and sensitive for detecting rpoB L378R (Gootenberg et al.2018 ; Kim et al.2020).Also, the structure of crRNA is crucial for the specificity of Cas12a-crRNA (Nguyen et al.2020 ; Ke et al.2021) It was found that cytosine residue bias, unchain temperature, purine content, and GC content at the PAM all affected the recognition efficiency and specificity of crRNA.According to previous studies, crRNAs sequences ranging from 28–35 bp were designed using the CRISPR website.CrRNA design was based on the requirement that PAM sequence and drug resistance mutation point must be placed in the proximal region of PAM (1–6 bp away from PAM) to improve the detection specificity of CRISPR-Cas12a.The mismatch base should be placed in the PAM-proximal seed region of 1–5 bp from PAM (Kim et al.2020 ; Swarts et al.2017).Studying the recognition specificity of non-5'TTTV3' PAM of LbCas12a or adding additional single base mutations can also effectively improve the CRISPR-Cas12a targeting recognition specificity. In the system, it is notable that ssDNA FAM-BHQ1 fluorescent reporter cleaved by collateral activity of Cas12a is used to generate the detection results visible by producing changes in the fluorescence signal of reaction system. the signal depends on incubated time, the concentrations of the reporters and RPA primers, and the ratio of crRNA to LbCas12a (Fig. 3 -Figure 6).We have found that 25 min incubation could maximize the detection activity of the CRISPR-Cas12a system, whereas one-pot detection of RPA amplification and CRISPR-Cas12a in a single reaction system is within 60 min(Ding et al.2020 ;Ding et al.2020).While fluorescence reporter concentrations had a strong dose effect on the direct visual signal, the high concentrations of the reporters might produce off-target effects (Fig. 4 a and b). Nonetheless, The CRISPR-Cas12A nucleic acid detection technology also has some limitations.For example, the PAM region limits its wide application, and the low concentration of fluorescent reporter also makes the fluorescence detection method unable to observe the results under natural light.It has been found that high concentration of Cas12a protein can allow more single base mismatches, and then affect the detection specificity of CRISPR-Cas detection system.A 6-base pairing of crRNA activates the "trans cleavage" activity of Cas12a, a smaller concentration of Cas12a and crRNA increases the minimum number of matching bases recognized by CRISPR-Cas. This system can be further improved in some aspects, including integrating an ultrabright fluorescent nanocable into the detection system that has a 1000-fold lower limitation of detection (Xie et al.2021), or optimizing the structure of crRNA, thereby reducing the amount of non-specific fluorescence reporters.In addition, since CRISPR-Cas12a-based detection only requires controlling the temperature of incubation and RPA amplification at 37 or 39°C, it is possible to use minimal equipment or water bath and ubiquitous smart phone technology to record and report the detection results (Ding et al.2020 ; Broughton et al.2020 ; Xie et al.2021 ; Yin et al.2019 ; Chen et al.2017). The reason that we choose the rpoB gene in this study is that the target of RIF is the β-subunit of bacterial DNA-dependent RNA polymerase, which is encoded by the rpoB gene.At the genetic level, Most RIF resistance is due to the accumulation of mutations within an 81-bp region of rpoB , termed the rifampicin resistance determinant region (RRDR).Mutations within this region account for up to 98% of the RIF resistance observed.The strong correlation between genotypic changes in this region resulting in phenotypic resistance makes the RRDR an optimal target for the design of rapid molecular diagnostics.Recent genomic studies identified several rpoB non-RRDR mutations that co-occurring with RRDR mutations in clinical isolates and may confer fitness compensation. rpoB non-RRDR mutations could be utilized as additional molecular markers for predicting the fitness of clinical rifampin-resistant M.tuberculosis strains, and the fitness cost of rifampicin-resistance mutations was attributable to their direct influence on RNAP activity. L378R mutation is the most common non-RRDR mutation. Notably, due to the lack of continuing horizontal gene transfer, many drug resistance phenotypes in M.tuberculosis are caused by chromosomal mutations.Rifampicin resistance primarily through rpoB mutations that results in alterations to the structure of the RIF-binding pocket and confer rifampicin resistance by decreasing the binding affinity of RIF to RNAP.In addition to resistance, rpoB mutations confer bacterial fitness costs directly by decreasing the transcriptional efficacy of RNAP or indirectly by altering genome-wide transcriptional profiles.RRDR mutations lead to a structural or surface electrostatic potential change in the catalytic center of RNAP, which may reduce transcriptional efficiency and bacterial fitness.Notably, most of these mutations are in loop regions.Loops are the most flexible parts of a protein, and mutations are in the loop regions could contribute to the modulation or diversification of protein functions.For RNAP, the rearrangement of the loop region near the active site plays an important role in its transcriptional activity. In clinical applications, it is critical to detect drug-resistant mutants in order to treat disease effectively.The detection system we developed presents a highly sensitive and specificity in detection of rpoB L378R as it did not have detectable off-target effects on other sequences (e.g., HEK293 T cell, M.smegmatis, M.aureus and E.coli , Fig. 8 C-E).This outcome may be attribute to the high recognition specificity of crRNA.In addition, the cost of a detection reaction is estimated at about 0.7 dollars and can be significantly decreased when scaled up for bulk production in our study.Furthermore, disposable microfluidics chips or lyophilized reagents might further improve and develop the CRISPR-Cas12a-based gene detection to enable simpler and higher-efficiency point-of-care in economically underdeveloped areas or outdoors (Li et al.2020 ; Song et al.2016 ; Song et al.2018).Moreover, this approach with the CRISPR-Cas12a system makes it possible to detect thousands or millions of nucleotide sequences, point mutations or pathogens with limited amounts of tested samples (Ackerman et al.2020 ; Vervoort et al.2017). Conclusion A simple, rapid, economical, highly specific, highly sensitive and visualized single-point mutations detection method was developed in this study based on CRISPR-Cas12a for the MTB drug-resistant gene rpoB L378R.This method establishes a solid foundation for developing a new detection kit to screen TB drug resistance gene mutations of RIF. Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose. Funding This work was supported by [Scientific Research Project of Ningxia Education Department] under Grant [No.NYG2024122]. Author Contribution Conceptualization, Yuma Yang and Li Yang ; methodology, Yuma Yang ; validation, Li Yang, Yue Zhu ; formal analysis, Yue Zhu and Haitao La ; investigation, Yuma Yang ; resources, Yanhui Yang ; data curation, Sihan Zhang and Xiumin Gu ; writing original draft preparation, Yuhan Wangand Yue Zhu ; writing review and editing, Yuma Yang ; visualization, Yuma Yang ; supervision, Shuming Zhang ; project administration, Hetian Lei ; funding acquisition, Yanhui Yang.All authors have read and agreed to the published version of the manuscript. Data availability statement The datasets used and analyzed during the current study are available from the corresponding authors on reasonable requests. 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Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7257131","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":498566699,"identity":"07936919-d413-4c92-ab06-0f6ecb69e758","order_by":0,"name":"Yuma Yang","email":"","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuma","middleName":"","lastName":"Yang","suffix":""},{"id":498566702,"identity":"b8f8bb02-ee68-4c3f-aef2-d0bbcdce7141","order_by":1,"name":"Li Yang","email":"","orcid":"","institution":"Ningxia Medical 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02:08:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7257131/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7257131/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89099451,"identity":"353fccfd-529c-4f1b-a591-0e19178db1a1","added_by":"auto","created_at":"2025-08-14 16:01:31","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":71836,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003edsDNA Cleavage of crRNA-guided LbCas12a\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003eSchematic of the components of CRISPR-Cas12a system and its cleavage principle.PAM: Protospacer Adjacent motif, dsDNA: double strand DNA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb.\u003c/strong\u003eThe profile of \u003cem\u003erpoB\u003c/em\u003e plasmid and sequencing result of \u003cem\u003erpoB \u003c/em\u003eL378R point mutation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec.\u003c/strong\u003eVerification of cleavage activity of CRISPR-Cas12a in \u003cem\u003erpoB \u003c/em\u003eL378R.M: DL5000 DNA marker, 1-4: \u003cem\u003erpoB\u003c/em\u003e_wild, \u003cem\u003erpoB\u003c/em\u003e_wild+CRISPR-Cas12a, \u003cem\u003erpoB \u003c/em\u003eL378R, \u003cem\u003erpoB \u003c/em\u003eL378R+CRISPR-Cas12a.The two cleavage products by LbCas12a under the crRNA guidance are 135 bp and 618 bp.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7257131/v1/a9e0e66ff7f78916f2c6ebf6.jpeg"},{"id":89099455,"identity":"d3290634-f31f-438f-8a07-9641703e952e","added_by":"auto","created_at":"2025-08-14 16:01:31","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":150324,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCRISPR-Cas12a-based nucleic acid detection was carried out by fluorescence signal readout\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003eSchematic of CRISPR-Cas12a-based fluorescence signal readout.RPA: Recombinase Polymerase Amplification, \u003cstrong\u003e+\u003c/strong\u003e: positive, \u003cstrong\u003e-\u003c/strong\u003e: negative.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb.\u003c/strong\u003eScreening result of RPA amplification primers.M: DL5000 marker, 1-6: different reactions with different RPA primer pairs, +: positive control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec.\u003c/strong\u003eVerification of eight reactions with different components based a combined system of CRISPR-Cas12a and ssDNA FAM-BHQ1.1-7: different reactions with different components, \u003cem\u003erpoB \u003c/em\u003eL378R and \u003cem\u003erpoB\u003c/em\u003e_wild type were added to reactions 3 and 7, respectively, PAGE: polyacrylamide gel electrophoresis.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7257131/v1/1ba1748737f2adc9f0a80a02.jpeg"},{"id":89101290,"identity":"d8271528-4954-47ef-bcee-a3fda867c112","added_by":"auto","created_at":"2025-08-14 16:17:32","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":139179,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOptimization of incubation time for fluorescence detection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003eDetermination of the best incubation time by observing fluorescence signal under UV light, LED blue light and no excitation light.W: \u003cem\u003erpoB\u003c/em\u003e_wild, Mut: \u003cem\u003erpoB \u003c/em\u003eL378R.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb.\u003c/strong\u003eReal-time fluorescence detection for \u003cem\u003erpoB\u003c/em\u003e_wild and \u003cem\u003erpoB \u003c/em\u003eL378R reactions with a multimode plate.*\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, **** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001, ns: not significant.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7257131/v1/f2d305e26c02f8067795f238.jpeg"},{"id":89100635,"identity":"b855ea9e-ff8e-47ae-87c7-9d0c7499f792","added_by":"auto","created_at":"2025-08-14 16:09:32","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":136378,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOptimization of concentrations of ssDNA FAM-BHQ1 reporter for fluorescence detection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003eDetermination of the best concentration of ssDNA FAM-BHQ1 reporter by observing fluorescence signal under UV light, LED blue light and no excitation light.W: \u003cem\u003erpoB\u003c/em\u003e_wild, Mut: \u003cem\u003erpoB \u003c/em\u003eL378R.1-4: different reactions with 1.6, 3.2, 4.8 and 6.4 μM ssDNA FAM-BHQ1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb.\u003c/strong\u003eReal-time fluorescence detection for \u003cem\u003erpoB\u003c/em\u003e_wild and rpoB L378R reactions with different concentration of ssDNA FAM-BHQ1 reporter by a multimode plate reader.*\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, **** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001, ns: not significant.\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7257131/v1/6605b739d3cde7605dc00bce.jpeg"},{"id":89099461,"identity":"811efe05-1a53-413d-bbb5-8f3762411d76","added_by":"auto","created_at":"2025-08-14 16:01:32","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":137156,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOptimization of concentrations of RPA primers for fluorescence detection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003eDetermination of the best concentration of RPA primers by observing fluorescence signal under UV light, LED blue light and no excitation light.W: \u003cem\u003erpoB\u003c/em\u003e_wild, Mut: \u003cem\u003erpoB \u003c/em\u003eL378R.1-4: different reactions with different molar mass of RPA primers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb.\u003c/strong\u003eReal-time fluorescence detection for \u003cem\u003erpoB\u003c/em\u003e_wild and \u003cem\u003erpoB \u003c/em\u003eL378R reactions with different concentration of RPA primers by multimode plate reader.*\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001, **** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001, ns: not significant.\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7257131/v1/735032bdeae31f581dac254e.jpeg"},{"id":89099453,"identity":"5f1fe685-2e8e-47ed-844d-6046add5399f","added_by":"auto","created_at":"2025-08-14 16:01:31","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":129445,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOptimization of the ratios of crRNA and LbCas12a for fluorescence detection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003eDetermination of the ratios of crRNA and LbCas12a by fluorescence intensity under UV light, LED blue light and no excitation light.1:1, 1:2 and 2:1 based on 400 nM of crRNA and LbCas12a.W: \u003cem\u003erpoB\u003c/em\u003e_wild, Mut: \u003cem\u003erpoB \u003c/em\u003eL378R.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb.\u003c/strong\u003eReal-time fluorescence detection for \u003cem\u003erpoB\u003c/em\u003e_wild and \u003cem\u003erpoB \u003c/em\u003eL378R reactions with different ratio of crRNA and LbCas12a by multimode plate reader.*\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001, **** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001, ns: not significant.\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7257131/v1/14dfdaeb55cffc2b291684fe.jpeg"},{"id":89100633,"identity":"cbef8261-c23c-4f90-b4c5-29c33b14a81f","added_by":"auto","created_at":"2025-08-14 16:09:32","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":72841,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic of lateral flow strip and its application for detecting rpoB L378R\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003eSchematic of lateral flow strip.+: positive, -: negative.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb.\u003c/strong\u003eEvaluation of detection effect by lateral flow strip.Error bars represent the means ± s.d.from replicates (n=3).Two-tailed Student \u003cem\u003et\u003c/em\u003e-test.*\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001, **** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001, ns: not significant.\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7257131/v1/eb417f37e4afcadf91ce348f.jpeg"},{"id":89099468,"identity":"1998dc3c-9892-4ecf-995a-8544a52b2d5d","added_by":"auto","created_at":"2025-08-14 16:01:32","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":266173,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetection of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eropB\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eL378R in biological samples with CRISPR-Cas12a\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea-b.\u003c/strong\u003eEvaluation of sensitivity and specificity in mixed system of \u003cem\u003eM.smegmatis\u003c/em\u003e,\u003cem\u003e M.aureus\u003c/em\u003e or \u003cem\u003eE.coli\u003c/em\u003e with \u003cem\u003erpoB\u003c/em\u003e_wild or \u003cem\u003erpoB \u003c/em\u003eL378R by fluorescence signal and lateral flow strip.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec.\u003c/strong\u003eEvaluation of RPA amplification specificity by nucleic acid electrophoresis.1-6: amplification reactions with H\u003csub\u003e2\u003c/sub\u003eO, HEK293T, MTB\u003cem\u003e H37Rv \u003c/em\u003e(\u003cem\u003erpoB \u003c/em\u003eL378R), MTB\u003cem\u003e H37Rv\u003c/em\u003e, \u003cem\u003eM.aureus\u003c/em\u003e and \u003cem\u003eM.smegmatis\u003c/em\u003e, respectively.M: DL1000 marker.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed.\u003c/strong\u003eAssessment of the detection specificity in MTB\u003cem\u003e H37Rv\u003c/em\u003e with \u003cem\u003erpoB \u003c/em\u003eL378R.1-6: different mixture referred to (\u003cstrong\u003eC\u003c/strong\u003e) by fluorescence intensity readout.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee.\u003c/strong\u003eSensitivity\u003cstrong\u003e \u003c/strong\u003eevaluation of the CRISPR-Cas12a system.Error bars represent the means ± s.d.from replicates (n=3).Two-tailed Student \u003cem\u003et\u003c/em\u003e-test.*\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, **** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001, ns: not significant.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-7257131/v1/4030dd790a7ec3685ec020e9.png"},{"id":89553993,"identity":"17fdc19c-de24-44b6-ad7e-dd270db2ba67","added_by":"auto","created_at":"2025-08-21 09:02:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2169528,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7257131/v1/516b007a-c5ea-4744-9ebf-57d0bfa28932.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Precision Detection of Rifampin-resistant rpoB L378R Mutation in Mycobacterium tuberculosis with CRISPR-Cas12a","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), remains a major global public health threat and a leading cause of death from an infectious agent(Sotgiu et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).According to the World Health Organization (WHO) Global Tuberculosis Report 2023, an estimated 10.6\u0026nbsp;million people fell ill with TB worldwide in 2022, and 1.3\u0026nbsp;million people died from the disease (Organization, 2024).While approximately 85% of drug-susceptible TB patients can be cured with a standard 6-month drug regimen, drug-resistant TB (DR-TB), particularly multidrug- or rifampicin-resistant TB (MDR/RR-TB) which affected an estimated 3.3% of new TB patients and 17% of previously treated patients in 2022, poses enormous challenges to TB control and treatment efforts(Organization, 2024) .The development of drug resistance is primarily driven by mutations in specific bacterial genes.Key mutations conferring resistance to first-line drugs include those in the rpoB gene (e.g.codons S450L, L430P, H445D/N/Y) for rifampicin (RIF) and in the katG gene (e.g., codon S315T) or promoter region for isoniazid (INH) (Ma et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Torres Ortiz et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Welekidan et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).Notably, the rpoB gene is the target for the widely used Xpert MTB/RIF assay, which detects RIF resistance by identifying mutations, primarily within the core RIF resistance-determining region (RRDR).Mutations like S450L and H445D/N/Y within the RRDR, as well as others like L378R and R871H outside this core region, are frequently associated with RIF resistance (Ma et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).To effectively combat TB, especially MDR-TB, there is a critical need for rapid, sensitive, and accurate diagnostic tools that can detect TB infection and drug resistance early, thereby simplifying diagnosis and reducing costs, particularly in resource-limited settings. Nucleic acid amplification tests (NAATs) have significantly advanced this goal.Their advantages in accuracy and speed compared to traditional smear microscopy have led the WHO to strongly recommend NAATs as the initial diagnostic test for all individuals with signs and symptoms of pulmonary TB, effectively replacing microscopy as the primary diagnostic standard in most settings(Organization, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, 2024).Polymerase chain reaction (PCR) is a foundational and widely used NAAT technology due to its high accuracy and specificity (Cao et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Radmard et al., 2019; Shragai et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).However, conventional PCR often requires sophisticated laboratory infrastructure, expensive equipment, and trained personnel.Other established NAATs include Xpert MTB/RIF (and Ultra), line probe assays (LPAs), and DNA sequencing (Cai et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Habte et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).Despite their utility, factors like cost, instrument dependency, and operational complexity can limit their accessibility in economically underdeveloped regions.To overcome these limitations, isothermal amplification methods such as recombinase polymerase amplification (RPA)(Ding et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Miao et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and loop-mediated isothermal amplification (LAMP) (Vanhomwegen et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) have been developed.These techniques operate at constant temperatures, potentially reducing reliance on complex thermocycling equipment.While RPA and LAMP have shown promise for detecting pathogens like ASFV and HBV(He et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Miao et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Vanhomwegen et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), their application for TB detection faces challenges, including achieving the necessary sensitivity and specificity in complex clinical samples, potential primer-dimer formation, and the need for robust and standardized protocols suitable for point-of-care use (He et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eNucleic acid detection can also be accomplished with CRISPR-Cas systems, in which many Cas proteins including Cas12a, Cas12b, Cas13a and Cas14 can recognize dsDNA, ssDNA or RNA sequence under the guidance of single guide (sg) RNA (Aquino-Jarquin, 2019; Bhattacharyya et al., 2018; Chen et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ding et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Gootenberg et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).CRISPR-Cas nucleic acid detection technology has been successfully applied for examining many diseases including Hepatitis B Virus (HBV)(Ding et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), SARS-CoV-2(Broughton et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Huang et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Joung et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), HIV(Ding et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), Dengue and ZIKA(Ackerman et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kaminski et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Myhrvold et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) ; in addition, CRISPR-Cas-based detection technologies have also achieved single nucleotide recognition(Kim et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Myhrvold et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).Therefore, CRISPR-Cas12a in combination with recombinase polymerase amplification (RPA) can be utilized to establish simple, fast, highly sensitive and specific detection methods for detecting the rifampin-resistant MTB strains with mutations such as rpoB_L378R(Kellner et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mustafa \u0026amp; Makhawi, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this study we hypothesized that the MTB drug resistance mutant gene rpoB_L378R could be used as a detection target, and the CRISPR-Cas12a system could detect single base differences in the mutant gene sequence.Our aim of this investigation was to establish a rapid, convenient, economical, highly specific, highly sensitive and visual detection method for MTB drug resistance point mutation by combining RPA isothermal amplification technology, fluorescence detection and paper chromatography.The result of this study lay the groundwork for the future development of MTB drug resistance mutation detection reagents for clinical applications.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003ch2\u003eMaterials\u003c/h2\u003e\n\u003cp\u003eMajor reagents involved in this study included EnGen LbCas12a (Cpf1) (New England BioLabs, M0653T), plasmid mini kit I (OMEGA, D6943-02), Luria broth (LB) media, Ampicillin (100 mg/mL), E.coli DH5\u0026alpha;competent cell, NEBuffer2.1, polyacrylamide gel electrophoresis Gel Fast Preparation Kit (EpiZyme, PG114), Gel Extraction Kit D2500 (OMEGA, D2500-02), TaKaRa MutanBEST Kit (Takara, R401), QuickCutTM Hind III (TAKARA, 1615), TwistAmp\u0026reg; Basic Kit (TwistDx, TABAS03KIT), Milenia HybriDtect1 (TwistDX, Milenia01), UltraPure DNase/RNase -Free Distilled Water (Thermo, 10977015) and RNase inhibitor (Takara, 2313A).Thermo Fisher Nanodrop 1000 Spectrophotom was used to quantify nucleic acid.Fluorescence signals were detected by PerkinElmer EnVision Multimode Plate Reader, and fluorescence imaging was gained by Bio-Rad GEL Doc XR.\u003c/p\u003e\n\u003ch2\u003e1.1 Methods\u003c/h2\u003e\n\u003ch3\u003eTarget sequence and crRNA preparation\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eWe selected\u003cem\u003e\u0026nbsp;rpoB\u003c/em\u003e gene sequence (from 760794 to 761540, long 753 bp) (GenBank accession NC_000962) as a research object.The\u0026nbsp;wild-type\u003cem\u003e\u0026nbsp;rpoB\u003c/em\u003e gene of MTB \u003cem\u003eH37Rv\u003c/em\u003e was downloaded from National Center for Biotechnology Information, and subsequently it was synthesized and cloned into topo plasmid (topo-\u003cem\u003erpoB\u003c/em\u003e) by Beijing Ruibiotech.Both ends of the \u003cem\u003erpoB\u003c/em\u003e gene were synthesized with restriction enzyme cutting sites for \u003cem\u003eHind\u0026nbsp;\u003c/em\u003eIII.To obtain \u003cem\u003erpoB\u003c/em\u003e_L378R (CTG\u0026gt;CGG) target sequence, the topo-\u003cem\u003erpoB\u003c/em\u003e plasmid was performed a point mutation in codon 378 with mutation primers (\u003cem\u003erpoB\u003c/em\u003eL378R_mut_F and \u003cem\u003erpoB\u003c/em\u003eL378R_mut_R) and TaKaRa MutanBEST Kit\u0026rsquo;s introduction.\u003c/p\u003e\n\u003cp\u003eThe crRNA was designed to pair with \u003cem\u003erpoB\u0026nbsp;\u003c/em\u003eL378R mutant sequence by CRISPOR website (http://crispor.tefor.net/crispor.py), and the principle of designing crRNA was described previously(Kellner et al.2019).All nucleic acid sequences that were mentioned above were synthesized by Beijing Ruibiotech.\u003c/p\u003e\n\u003ch3\u003eDesign of RPA amplification assay and primers\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eTo improve LbCas12a detection efficiency and sensitivity, RPA was performed with TwistAmp\u0026reg; Basic Kit.Four RPA primer pairs were designed at both ends of a mutant site of the \u003cem\u003erpoB\u0026nbsp;\u003c/em\u003eL378R\u0026nbsp;target sequence using Primer Premier 5.0 and refered to the TwistAmp Assay Design Manual.RPA primers length was designed from 19 nt to 35 nt, and the producing amplicon size was under 150 bp.Then, RPA primers were confirmed further by NCBI Primer-BLAST based on the RPA primers design principles.Before RPA assay, we selected the best primer pair based on the ratio and specificity of amplification, and then RPA amplification was performed strictly according to TwistAmp\u003csup\u003e\u0026reg;\u003c/sup\u003e Basic Kit\u0026rsquo;s introduction.The primer sequences were synthesized by Beijing Ruibiotech and\u0026nbsp;listed in Table 1.\u003c/p\u003e\n\u003ch3\u003eLbCas12a cleavage assay\u003c/h3\u003e\n\u003cp\u003eLbCas12a cleavage assay was carried out based on a previous protocol (Chen et al.2018 ;\u0026nbsp;Shi Y\u0026nbsp;et al.2022\u0026nbsp;).with slight modifications.Briefly, Cas12a cleavage reaction was implemented with 400 nM Cas12a, 400 nM \u003cem\u003erpoB\u0026nbsp;\u003c/em\u003eL378R-targeted crRNA, 1U/\u0026mu;L murine RNase inhibitor, and 100 ng \u003cem\u003erpoB\u0026nbsp;\u003c/em\u003eL378R or \u003cem\u003erpoB\u003c/em\u003e_wild type in 1 \u003cimg width=\"10\" height=\"21\" src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAAoAAAAVCAYAAAB/sn/zAAAAAXNSR0IArs4c6QAAAAlwSFlzAAAOxAAADsQBlSsOGwAAABl0RVh0U29mdHdhcmUATWljcm9zb2Z0IE9mZmljZX/tNXEAAAFoSURBVDhPY2lsbGQgBrAQowikZvgplHl8NuOi26T/wtcXzkQOhXTFO7Gr+OsMEv1UZoB9LSH+NXHLhm6zj6ld/5UO9cwCiZUE/UjrmMww85bG81sxDCrNYIVeda0TvzT0Lp0/uWEmb03X28mcJf/Lqy/PfGBawJCTbVs/uanpA1hhU8fOZXVNLax/q2onLe9IX5rz7TXDG/XYT+15nnlLOppWoAR4U9uKhXVdGe5Htb0jZ70KZli2Kmbdku6mhTA3w2OmriYucnlpqecTjYzfeap3GBZmTPOsWlgXubupaTncxLoa79A1dRVLFt9VYNKo7Y7qYS76X1y3efnEWoklpY11f7e2NK0Cm7i9oaF4+xsjJr2G4lyuDU3LmxgEGOpzREV65y6ZXNnAWWTDwgBR+OG90lLD+Lxl7zZMmAJz04RjKlPSAl4zndv3/D+DNDT13BYVnsyweypGipt7U3SSqfQTsPhQSGYAnbSP8g2RxTYAAAAASUVORK5CYII=\" alt=\"image\"\u003e\u0026nbsp;NEB buffer 2.1.This mixture was incubated at 37℃\u0026nbsp;for 60 min and then analyzed by 2% agarose gel electrophoresis.\u003c/p\u003e\n\u003ch3\u003eFAM-BHQ1 fluorescence reporter detection\u003c/h3\u003e\n\u003cp\u003eThe ssDNA FAM-BHQ1 reporters contained a poly (T) oligonucleotide sequence linked to fluorophore and quencher.In the FAM-BHQ1 fluorescence reporter detection assay, LbCas12a firstly bonded crRNA to form a LbCas12a-crRNA complex, and LbCas12a was activated when crRNA paired with the target DNA sequence, triggering the trans-cleavage activity of LbCas12a to cleave a non-target ssDNA reporter.In this assay there were 400 nM LbCas12a, 400 nM crRNA, 1 U/\u0026mu;L murine RNase inhibitor, 2.4 \u0026mu;M ssDNA FAM-BHQ1 fluorescent reporter and 100 ng target DNA \u003cem\u003erpoB\u0026nbsp;\u003c/em\u003eL378R or \u003cem\u003erpoB\u003c/em\u003e_wild type in 1 \u003cimg width=\"10\" height=\"21\" src=\"data:image/png;base64,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\" alt=\"image\"\u003e NEB buffer 2.1.The reaction mixtures were quickly transferred to a light-proof 96-well plate at Multimode Plate Reader preheated 37 ℃, and fluorescence signals were measured every 5 min (FAM:\u0026nbsp;\u0026lambda;\u003csub\u003eex\u003c/sub\u003e=492,\u0026nbsp;\u0026lambda;\u003csub\u003eem\u003c/sub\u003e=517).In addition, we also observed fluorescence intensity under light including UV, LED blue and no excitation light.In optimization of fluorescence detection conditions, we tried to improve fluorescent detection condition including incubating time (every 10 min for a total of 60 min), concentration of RPA primers (0.24, 0.36, 0.48, 0.60 \u0026mu;M) and ssDNA FAM-BHQ1 (1.6, 3.2, 4.8, 6.4 \u0026mu;M), and proportion of crRNA and Cas12a (1:1, 1:2, 2:1 based on 400 nM) to maximize detection effect.\u003c/p\u003e\n\u003ch3\u003eLateral flow readout of Cas12a detection\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eIn lateral flow detection assay, the reaction mixture contained 400 nM LbCas12a, 400 nM crRNA, 1 U/\u0026mu;L murine RNase inhibitor, 1 \u0026mu;M ssDNA FAM-biotin reporters, RPA reaction mix of \u003cem\u003erpoB\u0026nbsp;\u003c/em\u003eL378R or \u003cem\u003erpoB\u003c/em\u003e_wild and 1\u0026times;NEB buffer 2.1type.After incubation for 40 min, 100 \u0026mu;L of HybriDetect 1 assay buffer (from Milenia HybriDetect 1 kit) was added.Subsequently, lateral flow detection was performed by placing a HybriDetect 1 lateral flow strip into a reaction system.After 1-2 min the colored readout was developed.\u003c/p\u003e\n\u003ch3\u003ePreparation and analysis of the bacterial samples\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003erpoB\u003c/em\u003e-negative MTB \u003cem\u003eH37Rv\u003c/em\u003e strains were inactivated at 100℃ for 10 min, and the genome was extracted using a Bacterial DNA Kit and preserved at -80℃.1 nM of MTB \u003cem\u003eH37Rv\u003c/em\u003e genomic DNA, and 1 nM of \u003cem\u003erpoB\u0026nbsp;\u003c/em\u003eL378R fragments were mixed as \u003cem\u003erpoB\u003c/em\u003e-positive.Other controls included the genomic DNA of HEK293T cell, \u003cem\u003eM.smegmatis\u003c/em\u003e, \u003cem\u003eM.aureus\u003c/em\u003e and \u003cem\u003eE\u003c/em\u003e.\u003cem\u003ecoli\u003c/em\u003e.Noticeably, 2 \u0026mu;L of genomic DNA was subjected to RPA reaction and then to CRISPR-Cas12a detection in a fluorescence signal and lateral flow strip assay.\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Medical Ethics Review Committee of Ningxia Medical University, and the reference number is 2020-531.\u003c/p\u003e\n\u003ch3\u003eStatistical Analysis\u003c/h3\u003e\n\u003cp\u003eRepresentative data were the mean \u0026plusmn; standard deviation (SD) from triplicates.Statistical analyses were performed using GraphPad Prism 8.0.2 using two-way ANOVA.Significance was considered at *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 ; ns indicated no significance.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eCRISPR-Cas12a can be leveraged to detect a single nucleic acid mutation.We aimed to establish a rapid, convenient, economical, highly specific, highly sensitive and visual method for detecting MTB drug resistance point mutation.Thereby, we hypothesized that the CRISPR-Cas12a system could be used to detect MTB drug resistance mutant genes including \u003cem\u003erpoB\u0026nbsp;\u003c/em\u003eL378R.\u003c/p\u003e\n\u003ch2\u003edsDNA cleavage of \u003cem\u003eropB\u003c/em\u003eL378R based on CRISPR-Cas12a\u003c/h2\u003e\n\u003cp\u003eTo accomplish the CRISPR-Cas12a cleavage detection on dsDNA \u003cem\u003erpoB\u0026nbsp;\u003c/em\u003eL378R, as shown in Figure 1a, LbCas12a and crRNA were mixed to form a LbCas12a-crRNA complex at 37\u0026deg;C, the \u003cem\u003ecis\u003c/em\u003e- and \u003cem\u003etrans\u003c/em\u003e-acting cleavage activity of LbCas12a that could achieve targeted and no-targeted cleavage in turn was activated when crRNA and \u003cem\u003erpoB\u0026nbsp;\u003c/em\u003eL378R bases paired perfectly (Chen et al.2018).Here, \u003cem\u003erpoB-\u003c/em\u003eL378 was mutated to L378R (CTG\u0026gt;CGG) (Figure 1b).To test the \u003cem\u003ecis\u003c/em\u003e-cleavage activity of LbCas12a for \u003cem\u003erpoB\u0026nbsp;\u003c/em\u003eL378R compared to \u003cem\u003erpoB\u003c/em\u003e_wild type, mutant specific crRNA was designed to distinguish \u003cem\u003erpoB\u0026nbsp;\u003c/em\u003eL378R from \u003cem\u003erpoB\u003c/em\u003e_wild type (Figure 1c).\u003cem\u003eCis\u003c/em\u003e-acting cleavage lets a \u003cem\u003erpoB\u0026nbsp;\u003c/em\u003eL378R sequence (753 bp) divide into two sections shown by 2 % agarose gel electrophoresis, resulting in a shorter band only shown in lane 4 (Figure 1c).This shorter DNA fragment represented a positive result from the cleaved \u003cem\u003erpoB\u0026nbsp;\u003c/em\u003eL378R in the LbCas12a cleavage assay.\u003c/p\u003e\n\u003ch2\u003eDetection of \u003cem\u003erpoB\u0026nbsp;\u003c/em\u003eL378R with CRISPR-Cas12a is simple, rapid and specific\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eTo visually identify \u003cem\u003erpoB\u0026nbsp;\u003c/em\u003eL378R, LbCas12a trans-cleavage activity (collateral activity) was subjected to a non-targeted cleavage ssDNA FAM-BHQ1 fluorescence reporter assay (Figure 2a).In this assay, the RPA primers were first optimized.As shown in Figure 2b, there was a brighter and denser single band at about 150 bp in lane 6, indicating that this pair of RPA primers resulted in the best specificity and amplification efficiency among the tested pairs.Thereby, this pair of primers was used in the following experiments.We next verified LbCas12a trans-cleavage activity to a non-target DNA sequence (Figure 2a) and tested the specificity of point mutation discrimination by combining a ssDNA fluorescence reporter.As shown in Figure 2c, reaction 3 with \u003cem\u003erpoB\u0026nbsp;\u003c/em\u003eL378R sequence, crRNA and LbCas12a produced a stronger fluorescence signal than that with \u003cem\u003erpoB\u003c/em\u003e_wild in reaction 7, and a fluorescence signal could be directly seen under blue LED or UV light.In addition, the specificity of the generated fluorescence signal was confirmed by a polyacrylamide gel electrophoresis (PAGE) assay, as shown in Figure 2c.A thick stripe with shorter DNA size was in lane 3 (Figure 2c), representing the cleaved ssDNA FAM-BHQ1 reporters with a strong fluorescence signal.In other reaction conditions, there were only weak bands with relatively longer DNA sizes in corresponding lanes, which might be attributed to the fluorescence quench of the intact uncut reporter.These results indicate that LbCas12a detection assay provides a simple, rapid and specific detection of \u003cem\u003erpoB\u0026nbsp;\u003c/em\u003eL378R.\u003c/p\u003e\n\u003cp\u003eTo improve the CRISPR-Cas12a detection efficiency, we optimized fluorescence detection conditions.These included incubation time, the concentration of RPA primers and ssDNA FAM-BHQ1 fluorescence reporter, and the ratio of crRNA and LbCas12a.After identifying the mutants using the CRISPR-Cas12a fluorescence assay, we next tested if the incubation time of reaction could be reduced.The results (Figure 3a\u0026nbsp;and\u0026nbsp;b) showed that at the incubation time of 15-minutes (min) there was fluorescence under UV, LED blue light, and the strongest fluorescence was observed at the incubation time of 25 min.In optimizing concentrations of ssDNA FAM-BHQ1 reporters, the fluorescence could be observed under no excitation light in the reaction with 4.8 \u0026mu;M ssDNA FAM-BHQ1, and this concentration was used in the subsequent fluorescence assays (Figure 4a and b).In optimizing concentrations of RPA primers, we found that the primers at 0.24 \u0026mu;M gave the best result among the tested concentrations (Figure 5a and b).In particular, the fluorescence signal could be observed remarkably by naked eyes in Figure 5a.The ratio of crRNA to LbCas12a might play a critical role in improving cleavage activity and detection sensitivity.As shown in Figure 6a and b, at the ratios of 1:2 and 2:1 similar fluorescence signal emitted in real-time fluorescence curve, but we later observed that the ratio of 2:1 of crRNAs to LbCas12a gave a better result when crRNA was at 400 nM in the reaction system.\u003c/p\u003e\n\u003ch2\u003eDetection of \u003cem\u003eropB\u003c/em\u003eL378R with a CRISPR-Cas12a-original lateral flow detection assay\u003c/h2\u003e\n\u003cp\u003eTo reduce instrument dependence and increase efficiency, we combined lateral flow strip with CRISPR-Cas12a.In the lateral flow assay, 5\u0026rsquo;-FAM and 3\u0026rsquo;-biotin-labeled 12-nt ssDNA reporter were combined with a lateral flow strip for fluorescence intensity readout (Figure 7a) (Chen et al., 2018; Moreno-Mateos et al., 2017)Similar to the fluorescence detection assay, with lateral flow strips we quickly detected \u003cem\u003erpoB\u0026nbsp;\u003c/em\u003eL378R from \u003cem\u003erpoB\u003c/em\u003e_wild type with high sensitivity without requiring any signal detection instrument (Figure 7b).Band-intensity analysis showed a higher ratio of the test band to the control band resulting from the \u003cem\u003erpoB\u0026nbsp;\u003c/em\u003eL378R samples than from the \u003cem\u003erpoB\u003c/em\u003e_wild (Figure 7b).These results together show that there are two methods of fluorescence signal detection and paper strip systems to detect \u003cem\u003erpoB\u0026nbsp;\u003c/em\u003eL378R mutation with CRISPR-Cas12a, and these approaches are rapid, sensitive, and specific.\u003c/p\u003e\n\u003ch2\u003eDetection of \u003cem\u003erpoB\u0026nbsp;\u003c/em\u003eL378R in biological samples using CRISPR-Cas12a\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eNext, we evaluated the drug resistance mutation detection sensitivity and specificity with CRISPR-Cas12a-linked fluorescence detection and paper chromatography in different bacteria and human cells, which included HEK293T cell, \u003cem\u003eM.smegmatis, M.aureus,\u003c/em\u003e \u003cem\u003eE.coli,\u003c/em\u003e MTB\u003cem\u003e\u0026nbsp;H37Rv,\u003c/em\u003e and rifampicin-resistant MTB with\u003cem\u003e\u0026nbsp;rpoB\u0026nbsp;\u003c/em\u003eL378R.Firstly, \u003cem\u003erpoB\u003c/em\u003e_wild and \u003cem\u003erpoB\u0026nbsp;\u003c/em\u003eL378R fragments were mixed with \u003cem\u003eM.smegmatis,\u003c/em\u003e \u003cem\u003eM.aureus\u003c/em\u003e and \u003cem\u003eE.coli\u003c/em\u003e genomes at molar concentration 1:1, respectively.The CRISPR-Cas12a assay showed high specificity for \u003cem\u003erpoB\u0026nbsp;\u003c/em\u003eL378R (Figure 8a and b).Compared with the negative and wild-type control, the fluorescence intensity of \u003cem\u003erpoB\u0026nbsp;\u003c/em\u003eL378R reaction system was the strongest, and the difference of paper chromatography was highly significant (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001).In detection systems of H\u003csub\u003e2\u003c/sub\u003eO, HEK293T, MTB \u003cem\u003eH37Rv\u003c/em\u003e (\u003cem\u003erpoB\u0026nbsp;\u003c/em\u003eL378R), MTB \u003cem\u003eH37Rv,\u003c/em\u003e \u003cem\u003eM.smegmatis\u003c/em\u003e and \u003cem\u003eM.aureus,\u003c/em\u003e RPA effectively amplified \u003cem\u003erpoB\u003c/em\u003e sequence with specificity and efficiency (Figure 8c).Fluorescence signal and paper chromatography detections based on CRISPR-Cas12a were used to identify MTB H37Rv (\u003cem\u003erpoB\u0026nbsp;\u003c/em\u003eL378R).As is shown in the fluorescence detection, the fluorescence intensity of the \u003cem\u003erpoB\u0026nbsp;\u003c/em\u003eL378R reaction system was stronger than other reaction systems (Figure 8d).Moreover, the CRISPR-Cas12a system specifically recognized \u003cem\u003erpoB\u0026nbsp;\u003c/em\u003eL378R with a sensitivity of 100 aM, and there was also a significant difference in the stripe gray assay (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01) (Figure 8e)\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this report showed that CRISPR-Cas12a, together with RPA isothermal amplification can be utilized to detect the rifampin-resistant MTB strains with the mutations such as \u003cem\u003erpoB\u003c/em\u003e L378R.This approach we developed is rapid, convenient, economical, highly specific and sensitive for identifying \u003cem\u003erpoB\u003c/em\u003e L378R ; in addition, it did not need expensive equipment, and it is accurate because in this system the collateral activity of Cas12a enables highly specific and sensitive for detecting \u003cem\u003erpoB\u003c/em\u003e L378R (Gootenberg et al.2018 ; Kim et al.2020).Also, the structure of crRNA is crucial for the specificity of Cas12a-crRNA (Nguyen et al.2020 ; Ke et al.2021) It was found that cytosine residue bias, unchain temperature, purine content, and GC content at the PAM all affected the recognition efficiency and specificity of crRNA.According to previous studies, crRNAs sequences ranging from 28\u0026ndash;35 bp were designed using the CRISPR website.CrRNA design was based on the requirement that PAM sequence and drug resistance mutation point must be placed in the proximal region of PAM (1\u0026ndash;6 bp away from PAM) to improve the detection specificity of CRISPR-Cas12a.The mismatch base should be placed in the PAM-proximal seed region of 1\u0026ndash;5 bp from PAM (Kim et al.2020 ; Swarts et al.2017).Studying the recognition specificity of non-5'TTTV3' PAM of LbCas12a or adding additional single base mutations can also effectively improve the CRISPR-Cas12a targeting recognition specificity.\u003c/p\u003e\u003cp\u003eIn the system, it is notable that ssDNA FAM-BHQ1 fluorescent reporter cleaved by collateral activity of Cas12a is used to generate the detection results visible by producing changes in the fluorescence signal of reaction system. the signal depends on incubated time, the concentrations of the reporters and RPA primers, and the ratio of crRNA to LbCas12a (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-Figure 6).We have found that 25 min incubation could maximize the detection activity of the CRISPR-Cas12a system, whereas one-pot detection of RPA amplification and CRISPR-Cas12a in a single reaction system is within 60 min(Ding et al.2020 ;Ding et al.2020).While fluorescence reporter concentrations had a strong dose effect on the direct visual signal, the high concentrations of the reporters might produce off-target effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and b).\u003c/p\u003e\u003cp\u003eNonetheless, The CRISPR-Cas12A nucleic acid detection technology also has some limitations.For example, the PAM region limits its wide application, and the low concentration of fluorescent reporter also makes the fluorescence detection method unable to observe the results under natural light.It has been found that high concentration of Cas12a protein can allow more single base mismatches, and then affect the detection specificity of CRISPR-Cas detection system.A 6-base pairing of crRNA activates the \"trans cleavage\" activity of Cas12a, a smaller concentration of Cas12a and crRNA increases the minimum number of matching bases recognized by CRISPR-Cas.\u003c/p\u003e\u003cp\u003eThis system can be further improved in some aspects, including integrating an ultrabright fluorescent nanocable into the detection system that has a 1000-fold lower limitation of detection (Xie et al.2021), or optimizing the structure of crRNA, thereby reducing the amount of non-specific fluorescence reporters.In addition, since CRISPR-Cas12a-based detection only requires controlling the temperature of incubation and RPA amplification at 37 or 39\u0026deg;C, it is possible to use minimal equipment or water bath and ubiquitous smart phone technology to record and report the detection results (Ding et al.2020 ; Broughton et al.2020 ; Xie et al.2021 ; Yin et al.2019 ; Chen et al.2017).\u003c/p\u003e\u003cp\u003eThe reason that we choose the \u003cem\u003erpoB\u003c/em\u003e gene in this study is that the target of RIF is the β-subunit of bacterial DNA-dependent RNA polymerase, which is encoded by the \u003cem\u003erpoB\u003c/em\u003e gene.At the genetic level, Most RIF resistance is due to the accumulation of mutations within an 81-bp region of \u003cem\u003erpoB\u003c/em\u003e, termed the rifampicin resistance determinant region (RRDR).Mutations within this region account for up to 98% of the RIF resistance observed.The strong correlation between genotypic changes in this region resulting in phenotypic resistance makes the RRDR an optimal target for the design of rapid molecular diagnostics.Recent genomic studies identified several \u003cem\u003erpoB\u003c/em\u003e non-RRDR mutations that co-occurring with RRDR mutations in clinical isolates and may confer fitness compensation.\u003cem\u003erpoB\u003c/em\u003e non-RRDR mutations could be utilized as additional molecular markers for predicting the fitness of clinical rifampin-resistant \u003cem\u003eM.tuberculosis\u003c/em\u003e strains, and the fitness cost of rifampicin-resistance mutations was attributable to their direct influence on RNAP activity.\u003cem\u003eL378R\u003c/em\u003e mutation is the most common non-RRDR mutation.\u003c/p\u003e\u003cp\u003eNotably, due to the lack of continuing horizontal gene transfer, many drug resistance phenotypes in M.tuberculosis are caused by chromosomal mutations.Rifampicin resistance primarily through \u003cem\u003erpoB\u003c/em\u003e mutations that results in alterations to the structure of the RIF-binding pocket and confer rifampicin resistance by decreasing the binding affinity of RIF to RNAP.In addition to resistance, \u003cem\u003erpoB\u003c/em\u003e mutations confer bacterial fitness costs directly by decreasing the transcriptional efficacy of RNAP or indirectly by altering genome-wide transcriptional profiles.RRDR mutations lead to a structural or surface electrostatic potential change in the catalytic center of RNAP, which may reduce transcriptional efficiency and bacterial fitness.Notably, most of these mutations are in loop regions.Loops are the most flexible parts of a protein, and mutations are in the loop regions could contribute to the modulation or diversification of protein functions.For RNAP, the rearrangement of the loop region near the active site plays an important role in its transcriptional activity.\u003c/p\u003e\u003cp\u003eIn clinical applications, it is critical to detect drug-resistant mutants in order to treat disease effectively.The detection system we developed presents a highly sensitive and specificity in detection of \u003cem\u003erpoB\u003c/em\u003e L378R as it did not have detectable off-target effects on other sequences (e.g., HEK293 T cell, \u003cem\u003eM.smegmatis, M.aureus\u003c/em\u003e and \u003cem\u003eE.coli\u003c/em\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC-E).This outcome may be attribute to the high recognition specificity of crRNA.In addition, the cost of a detection reaction is estimated at about 0.7 dollars and can be significantly decreased when scaled up for bulk production in our study.Furthermore, disposable microfluidics chips or lyophilized reagents might further improve and develop the CRISPR-Cas12a-based gene detection to enable simpler and higher-efficiency point-of-care in economically underdeveloped areas or outdoors (Li et al.2020 ; Song et al.2016 ; Song et al.2018).Moreover, this approach with the CRISPR-Cas12a system makes it possible to detect thousands or millions of nucleotide sequences, point mutations or pathogens with limited amounts of tested samples (Ackerman et al.2020 ; Vervoort et al.2017).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eA simple, rapid, economical, highly specific, highly sensitive and visualized single-point mutations detection method was developed in this study based on CRISPR-Cas12a for the MTB drug-resistant gene \u003cem\u003erpoB\u003c/em\u003e L378R.This method establishes a solid foundation for developing a new detection kit to screen TB drug resistance gene mutations of RIF.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by [Scientific Research Project of Ningxia Education Department] under Grant [No.NYG2024122].\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, Yuma Yang and Li Yang ; methodology, Yuma Yang ; validation, Li Yang, Yue Zhu ; formal analysis, Yue Zhu and Haitao La ; investigation, Yuma Yang ; resources, Yanhui Yang ; data curation, Sihan Zhang and Xiumin Gu ; writing original draft preparation, Yuhan Wangand Yue Zhu ; writing review and editing, Yuma Yang ; visualization, Yuma Yang ; supervision, Shuming Zhang ; project administration, Hetian Lei ; funding acquisition, Yanhui Yang.All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eData availability statement\u003c/h2\u003e\u003cp\u003eThe datasets used and analyzed during the current study are available from the corresponding authors on reasonable requests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAckerman, C.M., Myhrvold, C., Thakku, S.G., Freije, C.A., Metsky, H.C., Yang, D.K., Ye, S.H., Boehm, C.K., Kosoko-Thoroddsen, T.F., Kehe, J., Nguyen, T.G., Carter, A., Kulesa, A., Barnes, J.R., Dugan, V.G., Hung, D.T., Blainey, P.C., \u0026amp; Sabeti, P.C.(2020).Massively 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version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"CRISPR-Cas12a, Mycobacterium tuberculosis, nucleic acid detection, rifampin, resistance, rpoB","lastPublishedDoi":"10.21203/rs.3.rs-7257131/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7257131/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRifampin is the most effective drug in the treatment of tuberculosis.However, certain strains of MTB have developed resistance to rifampin, leading to the need for alternative treatment options.The \u003cem\u003erpoB\u003c/em\u003e gene mutations play a central role in MTB resistance to the rifampin, so it is essential to identify these mutations and efficiently treat rifampin-resistant MTB strains.This study developed a novel CRISPR-Cas12a platform integrated with recombinase polymerase amplification (RPA) and fluorescence detection to specifically identify the \u003cem\u003erpoB\u003c/em\u003e_L378R mutation associated with Rifampin resistance in (MTB).We found that this detection system was highly specific and did not cross-react with created reference samples containing the genomes of MTB H37Rv, \u003cem\u003eMycobacterium smegmatis, Mycobacterium aureus\u003c/em\u003e, and \u003cem\u003eEscherichia coli.\u003c/em\u003eThe CRISPR-Cas12a-based platform developed in this study was simple, sensitive, and specific for detecting the Rifampin-resistant MTB strain with the \u003cem\u003erpoB\u003c/em\u003e_L378R mutation.This suggested that it has potential for clinical applications in identifying MTB \u003cem\u003erpoB\u003c/em\u003e_L378R mutation.\u003c/p\u003e","manuscriptTitle":"Precision Detection of Rifampin-resistant rpoB L378R Mutation in Mycobacterium tuberculosis with CRISPR-Cas12a","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-14 16:01:27","doi":"10.21203/rs.3.rs-7257131/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"74df5e03-3835-4ecd-b5b7-c5089af22735","owner":[],"postedDate":"August 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-21T08:54:05+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-14 16:01:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7257131","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7257131","identity":"rs-7257131","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}