Enhanced CRISPR/Cas-Based Immunoassay through Magnetic Proximity Extension and Detection DOI
Fangchi Shao, Jiumei Hu, Pengfei Zhang

и другие.

medRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Сен. 10, 2024

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-associated systems have recently emerged as a focal point for developing next-generation molecular diagnosis, particularly nucleic acid detection. However, the detection of proteins is equally critical across diverse applications in biology, medicine, and food industry, especially diagnosing prognosing diseases like cancer, Alzheimer's cardiovascular conditions. Despite recent efforts to adapt CRISPR/Cas protein with immunoassays, these methods typically achieved sensitivity only femtomolar picomolar range, underscoring need enhanced capabilities. To address this, we developed CRISPR-AMPED, an innovative CRISPR/Cas-based immunoassay by magnetic proximity extension This approach combines assay (PEA) beads that converts into DNA barcodes quantification effective washing steps minimize non-specific binding hybridization, therefore reducing background noise increasing sensitivity. The resulting are then detected through isothermal amplification testing (NAAT) using recombinase polymerase (RPA) coupled CRISPR/Cas12a system, replacing traditional PCR. integration eliminates thermocycling bulky equipment, reduces time, provides simultaneous target signal amplification, thereby significantly boosting CRISPR-AMPED achieves attomolar level sensitivity, surpassing ELISA over three orders magnitude outperforming existing systems. Additionally, our smartphone-based device demonstrates potential point-of-care applications, digital format extends dynamic range enhances quantitation precision. We believe represents significant advancement field

Язык: Английский

Multiplex Digital Nucleic Acid Analysis by a LAMP–Argonaute Coupling Assay via a Parallel Droplet Fusion SlipChip DOI

Yang Luo,

Xingyu Ye,

Nan Shen

и другие.

Analytical Chemistry, Год журнала: 2024, Номер 97(1), С. 731 - 740

Опубликована: Дек. 31, 2024

Multiplex digital nucleic acid analysis (NAA) allows the precise quantification of multiple target acids with single-molecule sensitivity, making it highly appealing for life science research and clinical diagnostics. Nucleic acid-guided endonucleases, such as CRISPR, have demonstrated great potential in NAA. However, performing multiplex NAA an endonuclease remains challenging. The thermophilic Argonaute protein (Ago) enables specific targeting sequences by a single enzyme, exhibiting superior detection. Here, we developed coupling amplification Ago-specific detection using parallel droplet fusion facilitated SlipChip. SlipChip can generate series droplets to perform loop-mediated isothermal (LAMP), followed containing Ago reagents mixing reactions, resulting three distinct fluorescence signals (FAM, ROX, Cy5) corresponding each sequence. We performed viral load respiratory viruses, including influenza A, B, SARS-CoV-2, within 60 min. In addition, used this LAMP-Ago assay analyze loads 34 samples. system provides capable high sensitivity specificity.

Язык: Английский

Процитировано

4
Appraisal of CRISPR Technology as an Innovative Screening to Therapeutic Toolkit for Genetic Disorders DOI
Ayesha Shahid,

Ambreen Zahra,

Sabin Aslam

и другие.

Molecular Biotechnology, Год журнала: 2025, Номер unknown

Опубликована: Фев. 2, 2025

Язык: Английский

Процитировано

0
CRISPR/Cas-powered “sweet” DNA nano-sponge for highly sensitive detection of pathogen nucleic acid with portable personal glucometer DOI

Huiling You,

Jingwen Shan,

Yang Bai

и другие.

Chemical Engineering Journal, Год журнала: 2025, Номер unknown, С. 160618 - 160618

Опубликована: Фев. 1, 2025

Язык: Английский

Процитировано

0
CRISPR Digital Sensing: From Micronano-Collaborative Chip to Biomolecular Detection DOI
Xinyi Wang, Longjie Zhong, Wenbin Zhang

и другие.

ACS Nano, Год журнала: 2025, Номер unknown

Опубликована: Май 24, 2025

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) sensing technology proved to be valuable during the COVID-19 pandemic through its sensitivity, specificity, robustness, and versatility. However, issues such as overreliance on amplification, susceptibility false positives, lack of quantification strategies, complex operation procedures have hindered broader application in bioanalysis clinical diagnostics. collision between micronano-collaborative chips CRISPR has effectively addressed these bottlenecks, offering innovative solutions for diagnosis treatment. Unlike conventional micronano chips, digital enhance CRISPR's response trace amounts target molecules by leveraging highly controllable local environments compartmentalized microreactors. This advancement improves detection efficiency revolutionizes traditional vitro bioanalytical processes. First, working principles, fabrication techniques, performance metrics CRISPR-based droplet microfluidics microarray are examined. Then, applications bioassays reviewed, emphasizing their importance advancing systems gene editing. Finally, prospects explored, particularly potential body surface biomonitoring development opportunities biomedical field.

Язык: Английский

Процитировано

0
Digitized Kinetic Analysis Enhances Genotyping Capacity of CRISPR-Based Biosensing DOI
Joon Soo Park, Patarajarin Akarapipad, Fan‐En Chen

и другие.

ACS Nano, Год журнала: 2024, Номер 18(27), С. 18058 - 18070

Опубликована: Июнь 26, 2024

CRISPR/Cas systems have been widely employed for nucleic acid biosensing and further advanced mutation detection by virtue of the sequence specificity crRNA. However, existing CRISPR-based genotyping methods are limited mismatch tolerance Cas effectors, necessitating a comprehensive screening crRNAs to effectively distinguish between wild-type point-mutated sequences. To circumvent limitation conventional genotyping, here, we introduce Single-Molecule kinetic Analysis via Real-Time digital CRISPR/Cas12a-assisted assay (SMART-dCRISPR). SMART-dCRISPR leverages differential kinetics signal increase in systems, which is modulated complementarity crRNA target sequence. It employs single-molecule measurements discern mutations based on profiles that could otherwise be obscured variations concentrations. We applied genotype notable SARS-CoV-2, point (K417N) deletion (69/70DEL), successfully distinguishing wild-type, Omicron BA.1, BA.2 SARS-CoV-2 strains from clinical nasopharyngeal/nasal swab samples. Additionally, introduced portable real-time sensing device streamline enhance its practicality point-of-care settings. The combination rapid sensitive isothermal with analysis format significantly enhances versatility genotyping.

Язык: Английский

Процитировано

2
Portable wide-field femtoliter-chamber imaging system for point-of-care digital bioanalysis DOI Creative Commons
Tatsuya Iida, Jun Ando,

Mami Yoshimura

и другие.

iScience, Год журнала: 2024, Номер 27(9), С. 110868 - 110868

Опубликована: Сен. 1, 2024

Recently, digital bioanalysis using femtoliter (fL)-chamber arrays has significantly improved the sensitivity, accuracy, and throughput of conventional nucleic acid antigen tests, with great potential for diagnosis infectious diseases underlying disorders. However, large size platforms costly assay consumables complicates its use in point-of-care testing (POCT). To solve these problems, this study, we developed a wide-field fL-chamber imaging system (COWFISH2), portable femtoliter-chamber (footprint: 14 × 22 cm), by redesigning various electronic controls optical systems COWFISH, accompanied development low-cost durable bioanalysis. As proof concept, was successfully performed hospital setting, amplification-free multiplex RNA detection SARS-CoV-2, influenza A virus, B virus. Collectively, COWFISH2 will facilitate versatile convenient POCT, contributing to improvement public health, including prevention diseases.

Язык: Английский

Процитировано

1
Early Detection of Wildlife Disease Pathogens Using CRISPR-Cas System Methods DOI
Adam A. Pérez, Guelaguetza Vázquez-Ortíz, Margaret E. Hunter

и другие.

The CRISPR Journal, Год журнала: 2024, Номер unknown

Опубликована: Окт. 31, 2024

Wildlife diseases are a considerable threat to human health, conservation, and the economy. Surveillance is critical component mitigate impact of animal in these sectors. To monitor diseases, CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein) biosensors have proven instrumental as diagnostic tools capable detecting unique DNA RNA sequences related their associated pathogens. However, despite significant advances general development biosensors, use support wildlife disease management lagging. In some cases, concern could be rapidly surveyed using with minimal technical, operational, or cost requirements end users. This review explores potential further leverage this technology advance monitoring highlights how concerted standardization protocols can help ensure data reliability.

Язык: Английский

Процитировано

1
Control of HSV-1 Infection: Directions for the Development of CRISPR/Cas-Based Therapeutics and Diagnostics DOI Open Access
Anastasiia O. Sosnovtseva, N. A. Demidova, Р. Р. Климова

и другие.

International Journal of Molecular Sciences, Год журнала: 2024, Номер 25(22), С. 12346 - 12346

Опубликована: Ноя. 17, 2024

It is estimated that nearly all individuals have been infected with herpesviruses, herpes simplex virus type 1 (HSV-1) representing the most prevalent virus. In cases, HSV-1 causes non-life-threatening skin damage in adults. However, patients compromised immune systems, it can cause serious diseases, including death. The situation further complicated by emergence of strains are resistant to both traditional and novel antiviral drugs. is, therefore, imperative new methods combating other herpesviruses be developed without delay. CRISPR/Cas systems may prove an effective means controlling herpesvirus infections. This review presents current understanding underlying molecular mechanisms infection discusses four potential applications fight against These include search for viral cellular genes serve as targets, optimization anti-HSV-1 activity vivo, development CRISPR/Cas-based diagnostics, validation drug resistance mutations.

Язык: Английский

Процитировано

1
Rapid and Portable Quantification of HIV RNA via a Smartphone-enabled Digital CRISPR Device and Deep Learning DOI Open Access
Hoan T. Ngo, Patarajarin Akarapipad, Pei‐Wei Lee

и другие.

medRxiv (Cold Spring Harbor Laboratory), Год журнала: 2023, Номер unknown

Опубликована: Май 16, 2023

For the 28.2 million people in world living with HIV/AIDS and receiving antiretroviral therapy, it is crucial to monitor their HIV viral loads ease. To this end, rapid portable diagnostic tools that can quantify RNA are critically needed. We report herein a quantitative digital CRISPR-assisted detection assay has been implemented within smartphone-based device as potential solution. Specifically, we first developed fluorescence-based reverse transcription recombinase polymerase amplification (RT-RPA)-CRISPR for isothermally rapidly detecting at 42 °C < 30 min. When realized commercial stamp-sized chip, yields strongly fluorescent reaction wells corresponding RNA. The isothermal condition strong fluorescence small chip unlock compact thermal optical components our device, allowing us engineer palm-size (70 × 115 80 mm) lightweight (< 0.6 kg) device. Further leveraging smartphone, wrote custom app control perform assay, acquire images throughout time. additionally trained verified Deep Learning-based algorithm analyzing wells. Using smartphone-enabled CRISPR were able detect 75 copies of 15 min demonstrate toward convenient monitoring combating epidemic.

Язык: Английский

Процитировано

1
CRISPR/Cas Systems for Enhancing Photosynthesis: Climate Resilience and Food Production DOI

Meena Barupal,

Ashwani Kumar

Опубликована: Янв. 1, 2024

To achieve the Sustainable Development Goal 2 (SDG 2) established by UN, there is a need to produce 15–20% more food from recent trends of production. Biotechnological advances in late twentieth century paved path for creation genetically engineered crop species enhance agricultural Recently programmable gene editing tools such as zinc-finger nucleases (ZFN), transcription activator-like effector (TALE) nucleases, clustered regularly interspaced short palindromic repeat (CRISPR) systems, and base editors have been developed edit any genome desirable traits. Furthermore, plant organelle genomes are distinct, encoding numerous genes crucial photosynthesis respiration. Any alteration photosynthetic functionality can directly impact Photosynthesis enzymes evolutionarily conserved green plants. There huge scope CRISPR genomics transform routine crops into superfoods. Chloroplast-targeting with magic cut allows engineering higher expression useful traits increased specific types oil content seeds or inclusion all essential vitamins plant-based diets. Knock-out knock-in genes, especially those related photosystem small subunits key CO2-fixing enzyme Rubisco (SSU-encoded rbcS) disease—resistance other intermediate metabolites plants facilitate high productivity even under climate change scenario. CRISPR-Cas9 enables multiple polyploid which remained challenging long time. Supercharging C3 advancement process production calories limited land area fertilizer. In this review, we address CRISPR-Cas9-based uses, challenges improve targeted efficiency

Язык: Английский

Процитировано

0