Methods For High Signal-To-Noise Imaging Of Chromosomal Loci In Cells Using Fluorescent Cas9

Patent Status

Brief Description

Cas9 is an endonuclease that binds complementary target DNA and generates site-specific breaks using two conserved nuclease domains. By inactivating both nuclease domains, dCas9 is produced, which functions as a programmable DNA binding protein. Current methods use dCas9-GFP fusions to image chromosomal loci, but have insufficient signal-to-noise ratio and often misidentify loci.

UC Berkeley researchers have engineered a Cas9 variant that can be labeled with small molecule fluorescent dyes. This variant utilizes a conformational change in Cas9 to provide highly specific identification of chromosomal loci, and has been shown to work in a proof-of-principle experiment using Förster resonance energy transfer (FRET) pairs.

Suggested uses

- Diagnostic tool to detect large-scale chromosomal alterations in vivo
- Identification of target sequence presence and copy number
- Research tool to screen candidate libraries 

Advantages

- High signal-to-noise imaging of chromosomal loci
- Can be used in vivo or in vitro
- Compatible with a variety of standard molecules used in imaging

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