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

Tech ID: 24759 / UC Case 2015-098-0

Patent Status

Country Type Number Dated Case
United States Of America Published Application 20180142222 05/24/2018 2015-098
European Patent Office Published Application 3307762 04/18/2018 2015-098
Patent Cooperation Treaty Published Application WO2016201138 12/15/2016 2015-098
 

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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Inventors

  • Doudna, Jennifer A.

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