CRISPR Diagnostics for Quantification and Rapid Diagnosis of Myotonic Dystrophy Type 1 Repeat Expansion Disorders

Repeat expansion disorders, exemplified by myotonic dystrophy
type
1 (DM1), present challenges in diagnostic quantification because of
the variability and complexity of repeat lengths. Traditional diagnostic
methods, including PCR and Southern blotting, exhibit limitations
in sensitivity and specificity, necessitating the development of innovative
approaches for precise and rapid diagnosis. Here, we introduce a CRISPR-based
diagnostic method, REPLICA (repeat-primed locating of inherited disease by Cas3), for
the quantification and rapid diagnosis of DM1. This method, using
in vitro-assembled CRISPR-Cas3, demonstrates superior sensitivity
and specificity in quantifying CTG repeat expansion lengths, correlated
with disease severity. We also validate the robustness and accuracy
of CRISPR diagnostics in quantitatively diagnosing DM1 using patient
genomes. Furthermore, we optimize a REPLICA-based assay for point-of-care-testing
using lateral flow test strips, facilitating rapid screening and detection.
In summary, REPLICA-based CRISPR diagnostics offer precise and rapid
detection of repeat expansion disorders, promising personalized treatment
strategies.