Fig. 1: Design and characterization of InCasor.
From: In vivo imaging of mitochondrial DNA mutations using an integrated nano Cas12a sensor
a Schematic of the integrated nano Cas12a sensor (InCasor) consisting of a DNA/Mg2+ hybrid nanoflower (DNF), Cas12a/crRNA and circular reporter (CLR) for mtDNA imaging in live cells. b Analysis of the divalent cation preference of Cas12a/crRNA using a fluorophore-quencher single-stranded DNA cleavage assay. n = 3, data show mean ± SD. c Design of multielement-encoded DNA template used for rolling circle amplification (RCA) to generate DNA/Mg2+ hybrid nanoflower (DNF). The complementary DNA template features 4 main elements: i) cell-targeting aptamer (Sgc8); ii) mitochondria-targeting aptamer (Cytochrome C aptamer); iii) circular reporter (CLR) binding element; iv) Cas12a/crRNA binding element. Phi 29 DNA polymerase produces long ssDNA with repeated copies of the complementary sequence of the template, which could envelop magnesium pyrophosphate generated during DNA synthesis to form a nanoflower structure. d Agarose gel electrophoresis analysis of InCasor preparation steps: Lane 1: DNA ladder, Lane 2: Template 1 (encoding Sgc8 aptamer and binding site for Cas12a/crRNA complex), Lane 3: Template 2 (encoding Cyt C apt and binding site for CLR, Lane 4: Primer 1, Lane 5: Primer 2, Lane 6: CLR, Lane 7: crRNA, Lane 8: DNF, Lane 9: DNF/CLR, Lane 10: InCasor. e The scanning transmission electron microscopy (STEM) and energy dispersive spectroscopy (EDS) mapping of InCasor probe. Scale bar: 100 nm. f, g InCasorND4 can recognize target mtDNA in vitro to generate a fluorescence signal (f) and be self-degraded (g). The experiment was independently repeated three times with similar results. Data are presented as mean ± SD. Source data from (b, d, and f) are provided as a Source Data file.
