Introduction for CRISPR LiveFISH and CASFISH Technology

2020-10-23

By snt_manager

Share to:

Genetic scissors: a tool for rewriting the code of life

Emmanuelle Charpentier and Jennifer A. Doudna have discovered one of gene technology’s sharpest tools: the CRISPR/Cas9 genetic scissors. Using these, researchers can change the DNA of animals, plants and microorganisms with extremely high precision. This technology has had a revolutionary impact on the life sciences, is contributing to new cancer therapies and may make the dream of curing inherited diseases come true.

Researchers need to modify genes in cells if they are to find out about life’s inner workings. This used to be time-consuming, difficult and sometimes impossible work. Using the CRISPR/Cas9 genetic scissors, it is now possible to change the code of life over the course of a few weeks.

What Is CRISPR?

CRISPR technology is a simple yet powerful tool for editing genomes. It allows researchers to easily alter DNA sequences and modify gene function. Its many potential applications include correcting genetic defects, treating and preventing the spread of diseases and improving crops.

In popular usage, “CRISPR” (pronounced “crisper”) is shorthand for “CRISPR-Cas9.” CRISPRs are specialized stretches of DNA. The protein Cas9 (or “CRISPR-associated”) is an enzyme that acts like a pair of molecular scissors, capable of cutting strands of DNA.

CRISPR LiveFISH

Researchers from Stanfordd University, Zhejiang University and Castilleja School report a robust, versatile approach named CRISPR Live-cell fluorescent in situ hybridization

(LiveFISH) using fluorescent oligos for genome tracking in broad cell types including primary cells.

Principle

The dCas9 protein and fluorescent labeled-sgRNA are assembled in vitro as fRNP (fluorescent ribonucleoproteins). ¹Chemically synthesized fluorescent gRNAs in complex with dCas proteins can facilitate rapid, robust, and scalable genomic DNA tracking and RNA imaging in living cells including primary cells. The target DNA-dependent protection of gRNAs within the Cas9:gRNA:DNA ternary complex in the RNase-rich environment enriches target signals while minimizing background noise.

CASFISH

Researchers from Howard Hughes Medical Institute,, University of California (Berkeley) and Albert Einstein College of Medicine report a novel approach that uses in vitro constituted nuclease deficient clustered regularly interspaced short palindromic repeats

(CRISPR)/CRISPR-associated caspase 9 (Cas9) complexes as probes to label sequence-specific genomic loci fluorescently without global DNA denaturation (Cas9-mediated fluorescence in situ hybridization, CASFISH).

Principle

²Using fluorescently labeled nuclease-deficient Cas9 (dCas9) protein assembled with various single-guide RNA (sgRNA), this technology demonstrated rapid and robust labeling of repetitive DNA elements in pericentromere, centromere, G-rich telomere, and coding gene loci. Assembling dCas9 with an array of sgRNAs tiling arbitrary target loci, this technology can be able to visualize nonrepetitive genomic sequences. The dCas9/sgRNA binary complex is stable and binds its target DNA with high affinity, allowing sequential or simultaneous probing of multiple targets. CASFISH assays using differently colored dCas9/sgRNA complexes allow multicolor labeling of target loci in cells. In addition, the CASFISH assay is remarkably rapid under optimal conditions and is applicable for detection in primary tissue sections. This rapid, robust, less disruptive, and cost-effective technology adds a valuable tool for basic research and genetic diagnosis.

References

1. Wang H, Nakamura M, Abbott T R, Zhao D, Luo K, Yu C, Nguyen C M, Lo A, Daley T P, La Russa M. CRISPR-mediated live imaging of genome editing and transcription [J]. Science, 2019, 365(6459): 1301-1305.

2. Deng W, Shi X, Tjian R, Lionnet T, Singer R H. CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells [J]. Proceedings of the National Academy of Sciences, 2015, 112(38): 11870-11875.

Today Celnovte 10th Anniversary！！！

Celnovte CE Mark Approved