Background

The development of genome editing technologies has enabled the creation of more accurate isogenic cell lines and animal models for the study of human disease and promotes exciting new possibilities for human gene therapy. Recent progress in developing programmable nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)–Cas-associated nucleases, has greatly accelerated the progress of gene editing from concept to clinical practice.

Although genome editing technologies are widely and efficiently applicable, eliminating uncertainties in genotype outcomes of edited lines has remained challenging. The current protocols to derive genome edited lines entail labor- and time-consuming processes that require the screening of a great number of clones as well as PCR and sequencing techniques to verify the desired clones.

Technology Overview

FAGE constitutes a robust method for streamlining the gene editing process by incorporating reporters to reduce hands on time by automating/simplifying the screening process.

The use of a fluorescent negative selection module allows screening and sorting out of undesired random/off site modified transformed cells whereas the use of a fluorescent positive selection module allows the identification of desired on-site modified cells.

The uniqueness of this technique lies in the usage of a composite of donors carrying different fluorescence in the positive selection module while targeting the same genomic sequence. This allows for the identification of biallelic modifications in one screening process.

Identification of the correctly transformed cells can be done by FACS or fluorescence imaging.

FAGE can be efficiently implemented for automated high-throughput genome editing, enabling fast identification of modified cells.

FAGE technology

Arias et al. (2017) Stem Cell Reports doi:10.1016/j.stemcr.2017.08.026. FACS-Assisted CRISPR-Cas9 Genome Editing Facilitates Parkinson’s Disease Modeling
Jarazo et al. (2019) Frontiers in Genetics. doi: 10.3389/fgene.2019.00190. Guidelines for Fluorescent Guided Biallelic HDR Targeting Selection With PiggyBac System Removal for Gene Editing

Benefits

Faster than conventional methods. Screening of positive clones can be performed using FACS analysis instead of PCR.
Multiple mutations can be generated in a high throughput manner creating single nucleotide variant collections.

Applications

Genome editing technology
Gene therapies

Opportunity

Licensing out

Fluorescence Assisted Genome Editing (FAGE)

Patents

IP Status

Seeking