Universal and high-fidelity DNA single nucleotide polymorphism detection based on a CRISPR/Cas12a biochip

Chemical Science, Год журнала: 2021, Номер 12(12), С. 4455 - 4462

Опубликована: Янв. 1, 2021

Single nucleotide polymorphisms (SNPs) are associated with many human diseases, so accurate and efficient SNP detection is of great significance for early diagnosis clinical prognosis. This report proposes a universal high-fidelity genotyping method in microfluidic point-of-care equipment based on the clustered regularly interspaced short palindromic repeat (CRISPR) system. Briefly, by systematically inserting protospacer-adjacent-motif (PAM) sequence, we improved universality CRISPR/Cas12a detection; removing complementary ssDNA introducing an additional mismatch, sensitivity specificity. We preloaded reagents into biochip automating process, increasing stability long-term storage. enables us to rapidly conveniently detect genotypes within 20 min. In practical application, successfully distinguished three (homozygous wild type; homozygous mutant heterozygous type) CYP1A1*2 (A4889G, rs1048943), CYP2C19*2 (G681A, rs4244285), CYP2C9*3 (A1075C, rs1057910), CYP2C19*3 (G636A, rs4986893) genes related multiple cancers from 17 blood samples. CRISPR/Cas12a-based method, being universal, accurate, sensitive, will have broad applications molecular diagnostics research.

Язык: Английский

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Signal amplification and output of CRISPR/Cas-based biosensing systems: A review

Analytica Chimica Acta, Год журнала: 2021, Номер 1185, С. 338882 - 338882

Опубликована: Июль 26, 2021

Язык: Английский

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Catalytic hairpin assembly as cascade nucleic acid circuits for fluorescent biosensor: Design, evolution and application

TrAC Trends in Analytical Chemistry, Год журнала: 2022, Номер 151, С. 116582 - 116582

Опубликована: Март 10, 2022

Язык: Английский

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Integrating CRISPR/Cas within isothermal amplification for point-of-Care Assay of nucleic acid

Talanta, Год журнала: 2022, Номер 243, С. 123388 - 123388

Опубликована: Март 12, 2022

Язык: Английский

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CRISPR-Cas12a-Empowered Electrochemical Biosensor for Rapid and Ultrasensitive Detection of SARS-CoV-2 Delta Variant

Nano-Micro Letters, Год журнала: 2022, Номер 14(1)

Опубликована: Авг. 4, 2022

Coronavirus disease 2019 (COVID-19) is a highly contagious caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The gold standard method for the diagnosis of SARS-CoV-2 depends on quantitative reverse transcription-polymerase chain reaction till now, which time-consuming and requires expensive instrumentation, confirmation variants relies further sequencing techniques. Herein, we first proposed robust technique-methodology electrochemical CRISPR sensing with advantages rapid, sensitivity specificity detection variant. To enhance capability, electrodes are uniformly decorated electro-deposited nanoparticles. Using DNA template identical to Delta spike gene sequence as model, our biosensor exhibits excellent analytical limit (50 fM) high linearity (R2 = 0.987) over six orders magnitude dynamic range from 100 fM 10 nM without any nucleic-acid-amplification assays. can be completed within 1 h stability benefits CRISPR-Cas system. Furthermore, based wireless micro-electrochemical platform, reveals promising application ability in point-of-care testing.

Язык: Английский

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Asymmetric CRISPR enabling cascade signal amplification for nucleic acid detection by competitive crRNA

Nature Communications, Год журнала: 2023, Номер 14(1)

Опубликована: Ноя. 18, 2023

Abstract Nucleic acid detection powered by CRISPR technology provides a rapid, sensitive, and deployable approach to molecular diagnostics. While exciting, there remain challenges limiting its practical applications, such as the need for pre-amplification lack of quantitative ability. Here, we develop an asymmetric assay cascade signal amplification nucleic acids leveraging trans -cleavage behavior competitive crRNA. We discover that reaction between full-sized crRNA split CRISPR-Cas12a can induce amplification, significantly improving target signal. In addition, find recognize fragmented RNA/DNA targets, enabling direct RNA Cas12a. Based on these findings, apply our quantitatively detect microRNA without pre-amplification, achieving sensitivity 856 aM. Moreover, using this method, analyze quantify miR-19a biomarker in plasma samples from bladder cancer patients. This has potential be widely applied simple sensitive various diagnostic settings.

Язык: Английский

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Targeting miRNA by CRISPR/Cas in cancer: advantages and challenges

Military Medical Research, Год журнала: 2023, Номер 10(1)

Опубликована: Июль 17, 2023

Clustered regulatory interspaced short palindromic repeats (CRISPR) has changed biomedical research and provided entirely new models to analyze every aspect of sciences during the last decade. In study cancer, CRISPR/CRISPR-associated protein (Cas) system opens avenues into issues that were once unknown in our knowledge noncoding genome, tumor heterogeneity, precision medicines. CRISPR/Cas-based gene-editing technology now allows for precise permanent targeting mutations provides an opportunity target small non-coding RNAs such as microRNAs (miRNAs). However, development effective safe cancer gene editing therapy is highly dependent on proper design be innocuous normal cells prevent introducing other abnormalities. This aims highlight cutting-edge approaches cancer-gene based CRISPR/Cas miRNAs therapy. Furthermore, we potential challenges CRISPR/Cas-mediated miRNA offer advanced strategies overcome them.

Язык: Английский

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Chemiluminescence Sensor for miRNA-21 Detection Based on CRISPR-Cas12a and Cation Exchange Reaction

Analytical Chemistry, Год журнала: 2023, Номер 95(6), С. 3332 - 3339

Опубликована: Янв. 30, 2023

Herein, a chemiluminescence (CL) biosensor based on CRISPR-Cas12a and cation exchange reaction was constructed to detect the biomarker microRNA-21 (miRNA-21). The rolling circle amplification (RCA) introduced convert each target RNA strand into long single-stranded DNA with repeated sequences, which acted as triggers initiate transcleavage activity of CRISPR-Cas12a. activated Cas12a could cleave biotinylated linker CuS nanoparticles (NPs) inhibit binding NPs streptavidin immobilized surface microplate, strongly reduced generation Cu2+ from between AgNO3, thus efficiently suppressed CL Cu2+-luminol-H2O2 system, giving "signal off" biosensor. With multiple amplification, detection limit developed sensor for miRNA-21 reached 16 aM. In addition, this is not only suitable professional instrument but also smartphone used tool purpose portable low-cost assay. This method be specifically quite low level in human serum samples various cancer cells, indicating its potential ultrasensitive molecular diagnostics.

Язык: Английский

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Cascade amplification strategy based on ultra-thin graphdiyne and CRISPR/Cas for real-time detection of tumor biomarker

Chemical Engineering Journal, Год журнала: 2023, Номер 466, С. 143230 - 143230

Опубликована: Апрель 29, 2023

Язык: Английский

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Highly Effective Detection of Exosomal miRNAs in Plasma Using Liposome-Mediated Transfection CRISPR/Cas13a

ACS Sensors, Год журнала: 2023, Номер 8(2), С. 565 - 575

Опубликована: Фев. 1, 2023

Exosomal miRNAs play a critical role in cancer biology and could be potential biomarkers for diagnosis. However, due to the low abundance of exosomes, recognizing detecting disease-associated exosomal an easy-to-operate way remain challenge. Herein, we used liposome-mediated membrane fusion strategy (MFS) transfect CRISPR/Cas13a into termed MFS-CRISPR, directly measuring plasma. Using MFS-CRISPR platform detection miR-21, achieve linear range spanning four orders magnitude (104-108 particles/mL) method is able detect miR-21 as 1.2 × 103 particles/mL. The MFS confine fluorescent signals fused vesicles, which can exosome heterogeneity analysis. Moreover, assay was evaluated by clinical samples, difference expression breast patients healthy donors significant. Because high sensitivity simplicity, proposed have promising diagnosis treatment monitoring.

Язык: Английский

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