CRISPR/Cas14a-Based Isothermal Amplification for Profiling Plant MicroRNAs.
Anal Chem
; 93(37): 12602-12608, 2021 09 21.
Article
in En
| MEDLINE
| ID: mdl-34506121
ABSTRACT
MicroRNAs (miRNAs) play key roles in biological processes in plants, such as stress resistance, yet can hardly be quantified by an enzyme-involved terminal polymerization process due to their 2'-O-methyl modifications at the 3'-terminal. Herein, we proposed a CRISPR/Cas14a-based rolling circle amplification (termed Cas14R) assay, allowing reverse transcription-free and demethylation-free detection of plant miRNAs with single-nucleotide resolution. The employment of target-templated rolling circle amplification circumvents the extension of the unaccessible 2'-O-methyl group at the 3'-terminal. Particularly, the activated Cas14a confers the trans-cleavage activity for identifying target single-stranded DNA sequences without the necessity of the protospacer adjacent motif, generalizing the detection of miRNA sequences and the integration of different isothermal amplification techniques. Ultimately, the Cas14R assay has been applied to profile miR156a to evaluate the ripeness process of banana, indicating its feasibility in analyzing the roles of miRNAs in biological processes of plants.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
MicroRNAs
Language:
En
Journal:
Anal Chem
Year:
2021
Document type:
Article
Affiliation country:
China