posted on 2024-01-26, 23:09authored byXinmiao Liu, Hao Yang, Jun Liu, Kerui Liu, Lulu Jin, Yong Zhang, Mohammad Rizwan Khan, Kai Zhong, Jijuan Cao, Qiang He, Xuhan Xia, Ruijie Deng
In situ profiling of single-nucleotide
variations
(SNVs) can elucidate drug-resistant genotypes with single-cell resolution.
The capacity to directly “see” genetic information is
crucial for investigating the relationship between mutated genes and
phenotypes. Fluorescence in situ hybridization serves
as a canonical tool for genetic imaging; however, it cannot detect
subtle sequence alteration including SNVs. Herein, we develop an in situ Cas12a-based amplification refractory mutation system-PCR
(ARMS-PCR) method that allows the visualization of SNVs related to
quinolone resistance inside cells. The capacity of discriminating
SNVs is enhanced by incorporating optimized mismatched bases in the
allele-specific primers, thus allowing to specifically amplify quinolone-resistant
related genes. After in situ ARMS-PCR, we employed
a modified Cas12a/CRISPR RNA to tag the amplicon, thereby enabling
specific binding of fluorophore-labeled DNA probes. The method allows
to precisely quantify quinolone-resistant Salmonella
enterica in the bacterial mixture. Utilizing this
method, we investigated the survival competition capacity of quinolone-resistant
and quinolone-sensitive bacteria toward antimicrobial peptides and
indicated the enrichment of quinolone-resistant bacteria under colistin
sulfate stress. The in situ Cas12a-based ARMS-PCR
method holds the potential for profiling cellular phenotypes and gene
regulation with single-nucleotide resolution at the single-cell level.