CRISPR-Based Split Luciferase as a Biosensor for Unique DNA Sequences In Situ

Area of Science:

• Molecular Biology
• Biotechnology
• Genomics

Background:

• Current CRISPR-based DNA targeting primarily uses fluorescent reporters with catalytically inactive Cas9 (dCas9) for imaging, not direct detection of genomic DNA.
• There is a need for sensitive, single-cell resolution methods to directly detect endogenous genomic DNA sequences.

Purpose of the Study:

• To describe a protocol for a novel DNA biosensing approach for noninvasive, in situ detection of user-defined DNA sequences.
• To enable sensitive detection of single DNA copies within individual living cells.

Main Methods:

• Utilized a dual fusion protein biosensor comprising two NanoLuc luciferase (NLuc) fragments fused to dCas9.
• Paired the dCas9 fusion protein with user-defined single-guide RNAs (sgRNAs) for specific DNA targeting.
• Employed standard laboratory equipment like microscopes and luminescence microplate readers for detection.

Main Results:

• Demonstrated the capability to sensitively detect unique copies of a target DNA sequence.
• Achieved single-cell resolution for DNA detection.
• The biosensor functions noninvasively within living cells (in situ).

Conclusions:

• The developed DNA biosensor offers a promising alternative for direct, sensitive detection of endogenous genomic DNA.
• This method allows for user-defined sequence targeting with high specificity and resolution.
• The protocol facilitates the use of common laboratory equipment for advanced DNA analysis in living cells.