An Ultrasensitive, One-Pot RNA Detection Method Based on Rationally Engineered Cas9 Nickase-Assisted Isothermal Amplification Reaction

Autor:	Ting Wang, Yan Wang, Pinru Chen, Bin-Cheng Yin, Bang-Ce Ye
Rok vydání:	2022
Předmět:	Salmonella typhimurium SARS-CoV-2 RNA Ribosomal 16S Escherichia coli Deoxyribonuclease I HIV RNA Viral DNA CRISPR-Cas Systems Analytical Chemistry
Zdroj:	Analytical Chemistry. 94:12461-12471
ISSN:	1520-6882 0003-2700
Popis:	RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR) have revolutionized molecular diagnostics by offering versatile Cas effectors. We previously developed an isothermal amplification reaction method using Cas9 nickase (Cas9 nAR) to detect genomic DNA. However, slow dissociation of Cas9n from nicked double-stranded DNA (dsDNA) substrates dramatically hampers the cooperation between Cas9n and DNA polymerase, leading to low amplification efficiency. Here, we use structure-guided protein engineering to generate a Cas9n variant with faster kinetics and enhanced targeting specificity, and apply it to develop Cas9 nAR version 2 (Cas9 nAR-v2) by deftly merging reverse transcription with nicking-extension-displacement-based amplification for isothermal, one-pot RNA detection. This assay is validated by detecting
Databáze:	OpenAIRE
Externí odkaz:	https://explore.openaire.eu/search/publication?articleId=doi_dedup___::2a9ba1e0705899784513211890a4b283 https://doi.org/10.1021/acs.analchem.2c02617 Zobrazit plný text záznamu