Have you ever wanted to detect very small fragments of genomic DNA but there were no effective tools available? Our novel method based on the CRISPR-Cas9 system and T7 exonuclease, named CRISPR-ExoFISH, now allows to detect genomic loci as small as 2 kb!
The idea behind DNA FISH
DNA fluorescence in situ hybridization (FISH) is a powerful laboratory technique that has been used in diagnostics and genetic research. In this approach, chromosomes are exposed to a small DNA sequence - a probe that has a fluorescent molecule attached to it. The fluorescently labeled probe binds to a specific DNA sequence on a chromosome, allowing to study chromatin structure
and chromosomal abnormalities.
Limitations of traditional DNA FISH assays
Traditional Bacterial Artificial Chromosome (BAC) or oligonucleotide based probes to detect DNA in situ need the genomic DNA to be opened by heat denaturation and is only effective for targets of a certain length.
Firstly, heat denaturation step might affect the fine structure of nuclei, limiting chromatin study. Secondly, BAC-based DNA FISH probes usually spans over 150 kilobases (kb) DNA fragments
to generate sufficient hybridization signal for detection. Necessity of a relatively long target region can be restricting for some fields of study. For example, in cell and gene therapy exogenous gene introduced into the genome is usually less than 10 kb, making it impossible to detect and locate using traditional DNA FISH assays.
Novel design for improved detection of small DNA fragments
To overcome limitations of traditional method we envisioned a novel approach - CRISPR-ExoFISH, which combines our HuluFISH probes with CRISPR-Cas9 system and T7 exonuclease (bioRxiv preprint).
The Cas9 nickase-sgRNA ribonucleoprotein complex opens the genome loci at non-targeting strand (NTS) while T7 exonuclease finds the flapped DNA on NTS and digests it. This results in a single strand DNA (ssDNA) fragment, allowing access for HuluFISH probe. For the following hybridization, heat denaturation is not needed and a region below 5 kb is sufficient for the probe binding and generating a specific signal.
This novel method of detecting very small DNA fragments opens the door for many applications
in the fields like quantifying vector copy number (VCN) in cell and gene therapy (CGT), studying fine chromatin structure or validating knock-out/in fragment in cell lines and tissue.
Let us know in the comments, what are your thoughts on traditional DNA FISH restraints
and possible ways you would like to apply CRISPR-ExoFISH.