CASMART, a one-step CRISPR Cas12a-mediated isothermal amplification for rapid and high-resolution digital detection of rare mutant alleles.
Biosens Bioelectron
; 222: 114956, 2023 Feb 15.
Article
em En
| MEDLINE
| ID: mdl-36525708
Convenient, ultrasensitive, and accurate detection of rare variants is essential for early cancer diagnosis and precision medicine, however, despite years of efforts, tools that have all these qualities remain elusive. Here, we developed a one-step CRISPR/Cas12a-based digital diagnostic platform for accurately quantifying mutant alleles, referred to as the CRISPR ASsoaciated Mutation Allele Rapid Test (CASMART). The platform accurately quantifies the variant allele frequency of EGFR L858R within 1 h at 42 °C and can detect mutant targets as low as 0.3 copies/µL (0.498 aM) in mock multiplex cfDNA samples. We further investigated the applicability of CASMART using human genomic samples with confirmed EGFR L858R mutations previously measured variant allele frequency by next-generation sequencing. Comparison across platforms revealed equivalent detection performance (Pearson's correlation coefficient, R2 = 0.9208) and high quantification accuracy for mutation allele frequency (intraclass correlation coefficient = 0.959). Our one-step approach enables easy and accurate variant allele frequency measurement of rare mutant alleles without PCR instrumentation, while the assay time was reduced by approximately half compared to the digital PCR with the shortest turnaround. The CASMART is an alternative to conventional single nucleotide polymorphism detection methods with great potential as a next-generation biosensor for rapidly quantifying the variant allele fraction, especially in resource-limited settings.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Técnicas Biossensoriais
/
Sistemas CRISPR-Cas
Tipo de estudo:
Diagnostic_studies
Limite:
Humans
Idioma:
En
Revista:
Biosens Bioelectron
Assunto da revista:
BIOTECNOLOGIA
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
China