Abstract
To date, CRISPR-based DNA targeting approaches have typically used fusion proteins between full fluorescent reporters and catalytically inactive Cas9 (dCas9) for imaging rather than detection of endogenous genomic DNA sequences. A promising alternative strategy for DNA targeting is the direct biosensing of user-defined sequences at single copy with single-cell resolution. Our recently described DNA biosensing approach using a dual fusion protein biosensor comprised of two independently optimized fragments of NanoLuc luciferase (NLuc) directionally fused to dCas9 paired with user-defined single-guide RNAs (sgRNAs) could allow users to sensitively detect unique copies of a target sequence in individual living cells using common laboratory equipment such as a microscope or a luminescence-equipped microplate reader. Here we describe a protocol for using such a DNA biosensor noninvasively in situ.
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Heath, N.G., Segal, D.J. (2024). CRISPR-Based Split Luciferase as a Biosensor for Unique DNA Sequences In Situ. In: Haimovich, G. (eds) Fluorescence In Situ Hybridization (FISH). Methods in Molecular Biology, vol 2784. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3766-1_19
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DOI: https://doi.org/10.1007/978-1-0716-3766-1_19
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