Cas12a-based one-pot SNP detection with high accuracy

Zhang, Hongxia; zhang, Caixiang; Lu, Shuhan; Tong, Xiaohan; Zhang, Kun; Yin, Hao; Zhang, Ying

doi:10.1016/j.cellin.2023.100080

Cited by 16 publications

(12 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…6 A-B)( J. S. Chen et al., 2018 ; Ding et al., 2020 ; Gootenberg et al., 2018 ; Joung et al., 2020 ; Lu et al., 2020 ; Lu et al., 2022 ; Teng et al., 2019 ; H. X. Zhang et al., 2023 ). The detection limit for two-steps reaction using CtCas12a reached 200 ag per reaction for DNA substrate containing EV71 sequence, equivalent to～6.5 copies/uL ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…For application of nucleic acids detection, Cas effector is usually applied after target amplification, and such tolerance would not often cause false positive outcomes due to enrichment of its target sequences by isothermal amplification ( J. S. Chen et al., 2018 ; Ding et al., 2020 ; Gootenberg et al., 2018 ; Gootenberg et al., 2017 ; Joung et al., 2020 ; Lu et al., 2020 ; Lu et al., 2022 ; Teng et al., 2019 ; H. X. Zhang et al., 2023 ). In addition, it is worth to explore the potential off-target editing outcomes of CtCas12a in human cells.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, Cas12a holds activities of trans -cleavage of single-stranded DNA (ssDNA) after acting on dsDNA, allowing it usage in molecular diagnosis in combination with isothermal amplification technology such as recombinase polymerase amplification (RPA) or loop-mediated isothermal amplification (LAMP) ( J. S. Chen et al., 2018 ; Ding et al., 2020 ; Gootenberg et al., 2017 ; Joung et al., 2020 ; Lu et al., 2020 ; Lu et al., 2022 ; Teng et al., 2019 ; H. X. Zhang et al., 2023 ). However, owing to the restriction of PAM and reaction temperature, the usage of Cas12a is limited.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Characterization of a thermostable Cas12a ortholog

Wu,

Gao,

Shi

et al. 2023

Cell Insight

View full text Add to dashboard Cite

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Characterization of a thermostable Cas12a ortholog

Wu,

Gao,

Shi

et al. 2023

Cell Insight

View full text Add to dashboard Cite

“…[4] Previous reports have suggested that sensitivity reductions in one-pot systems are a result of the exonuclease activity (ciscleavage) of the Cas-based ribonucleoprotein (RNP), which hydrolyses target DNA and hinders efficient nucleic acid amplification. [5] Based on this hypothesis, we decided to explore whether deliberate modulation of the functional concentration of the Cas-based RNP at the start of the assay could be leveraged to control exonuclease activity and ultimately lead to improved signal generation in one-pot RPA-CRISPR-Cas12a assays. We evaluate the individual aspects of this approach, including the kinetics of RNP formation, cis exonuclease activity, and the diffusion of key reaction components to develop a mathematical model for determining optimal cis cleavage rates in RPA-CRISPR-Cas12 assays.…”

Section: Introductionmentioning

confidence: 99%

“…Previous studies have suggested that at low nucleic acid concentrations, target cleavage can outcompete target amplification, thus hamstringing the entire signal generation cascade. [5,11,12] The simplest methods for minimizing this competition involve initially separating the CRISPR and NAAT reactions within the same vessel, and then combining them after a defined period. A popular way to achieve this is to store the CRISPR-Cas reaction components in the lid of a reaction tube, and then perform an NAAT reaction in the bottom of the same tube.…”

Section: Introductionmentioning

confidence: 99%

In situ complexation of sgRNA and Cas12a improves the performance of a one-pot RPA–CRISPR-Cas12 assay

Lesinski,

Moragues,

Mathur

et al. 2024

Preprint

View full text Add to dashboard Cite

Due to their ability to selectively target pathogen-specific nucleic acids, CRISPR-Cas systems are increasingly being employed as diagnostic tools. “One pot” assays that combine nucleic acid amplification and CRISPR-Cas systems (NAAT–CRISPR-Cas) in a single step have emerged as one of the most popular CRISPR-Cas biosensing formats. However, operational simplicity comes at a cost, with one-pot assays typically being less sensitive than corresponding two-step NAAT–CRISPR-Cas assays and often failing to detect targets at low concentrations. It is thought that these performance reductions result from the competition between the two enzymatic processes driving the assay, namely Cas-mediated cis-cleavage and polymerase-mediated amplification of the target DNA. Herein, we describe a novel one-pot RPA–Cas12a assay that circumvents this issue by leveraging in situ complexation of the target-specific sgRNA and Cas12a to purposefully limit the concentration of active Cas12a during the early stages of the assay. Using a clinically relevant assay against a DNA target for HPV-16, we show how this in situ format reduces competition between target cleavage and amplification and engenders significant improvements in detection limit when compared to the traditional one-pot assay format, even in patient-derived samples. Finally, to gain further insight into the assay, we use experimental data to formulate a mechanistic model describing the competition between the Cas enzyme and nucleic acid amplification. These findings suggest that purposefully limiting cis-cleavage rates of Cas proteins is a viable strategy for improving the performance of one-pot NAAT–CRISPR-Cas assays.

show abstract

Development of a rapid detection assay for acetolactate synthase inhibitors resistance in three Amaranthus weed species through loop‐mediated isothermal amplification

Milani,

Panozzo,

Grazia

et al. 2024

J Sci Food Agric

View full text Add to dashboard Cite

BACKGROUNDThe early detection of herbicide resistance in weeds is a key factor to avoid herbicide waste and improve agriculture sustainability. The aim of this work was to develop and validate an allele‐specific loop‐mediated isothermal amplification (AS‐LAMP) assay for the quick on‐site detection of the resistance‐endowing point mutation Trp‐574‐Leu in the acetolactate synthase (ALS) gene in three widely diffused Amaranthus weed species, A. retroflexus, A. hybridus and A. tuberculatus.RESULTSThe AS‐LAMP protocol was developed on wild type and ALS‐mutant plants of the three species and revealed that the amplification approach with only the primer set specific for the mutant allele (574‐Leu) was the most promising. The validation and estimation of the AS‐LAMP performance evaluated by comparing the results with those of the molecular marker (CAPS, cleaved amplified polymorphic sequences) indicated that while sensitivity and specificity resulted relatively high in all species (overall 100% and >65%, respectively), precision was high for Amaranthus hybridus L. and Amaranthus retroflexus L. (75% and 79%, respectively), but quite low for Amaranthus tuberculatus (Moq.) J. D. Sauer (59%). The LAMP assay was also effective on crude genomic DNA extraction, allowing the quick detection of mutant plants in field situation (on site resistance detection).CONCLUSIONSThe proposed AS‐LAMP method has proven to be a promising technique for rapid detection of resistance due to Trp‐574‐Leu on the two monoecious weedy Amaranthus species but resulted less effective in the genetically variable dioecious species A. tuberculatus.This article is protected by copyright. All rights reserved.

show abstract

Cas12a-based one-pot SNP detection with high accuracy

Cited by 16 publications

References 30 publications

Characterization of a thermostable Cas12a ortholog

Characterization of a thermostable Cas12a ortholog

In situ complexation of sgRNA and Cas12a improves the performance of a one-pot RPA–CRISPR-Cas12 assay

Development of a rapid detection assay for acetolactate synthase inhibitors resistance in three Amaranthus weed species through loop‐mediated isothermal amplification

Contact Info

Product

Resources

About