Gene editing: not just for translation anymore

  title={Gene editing: not just for translation anymore},
  author={Moira A. McMahon and Meghdad Rahdar and Matthew H. Porteus},
  journal={Nature Methods},
Engineered nucleases have advanced the field of gene therapy with the promise of targeted genome modification as a treatment for human diseases. Here we discuss why engineered nucleases are an exciting research tool for gene editing and consider their applications to a range of biological questions. 

Special Issue: Manifesting Synthetic Biology

The existing assays for quantifying on- and off-target genome editing outcomes are reviewed and their utility in advancing the technology is described and their importance for the genome editing field is discussed.

Quantifying on- and off-target genome editing.

Key to Delivery: The (Epi-)genome Editing Vector Toolbox.

Current viral and non-viral delivery approaches applicable for genome and epigenome editing are summarized and discussed and their respective advantages and limitations are discussed.

Improvements in Gene Editing Technology Boost Its Applications in Livestock

The methods developed to improve efficiency and specificity of gene editing tools as well as approaches that can be employed for gene regulation, base editing, and epigenetic modifications are discussed.

Assembly and characterization of megaTALs for hyperspecific genome engineering applications.

This chapter describes the process of assembling a megaTAL from a meganuclease, as well as a method for characterization of nuclease cleavage activity in vivo using a fluorescence reporter assay.

Applications of CRISPR/Cas9 for Gene Editing in Hereditary Movement Disorders

The applicability of CRISPR/Cas9 to preclinical studies or gene therapy in hereditary movement disorders is discussed and advantages in terms of clinical applicability over other genome editing technologies such as zinc-finger nucleases and transcription activator-like effector nucleases are discussed.

megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering

The development of a single-chain rare-cleaving nuclease architecture is described, which is designated ‘megaTAL’, in which the DNA binding region of a transcription activator-like (TAL) effector is used to ‘address’ a site-specific meganuclease adjacent to a single desired genomic target site.

Quantifying genome-editing outcomes at endogenous loci with SMRT sequencing.

Chemically Modified Cpf1-CRISPR RNAs Mediate Efficient Genome Editing in Mammalian Cells.




A TALE nuclease architecture for efficient genome editing

This study identifies TALE truncation variants that efficiently cleave DNA when linked to the catalytic domain of FokI and uses them to generate discrete edits or small deletions within endogenous human NTF3 and CCR5 genes at efficiencies of up to 25%.

Genome editing with engineered zinc finger nucleases

A broad range of outcomes has resulted from the application of the same core technology: targeted genome cleavage by engineered, sequence-specific zinc finger nucleases followed by gene modification during subsequent repair.

High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases

Methods for using simple ssDNA oligonucleotides in tandem with ZFNs to efficiently produce human cell lines with three distinct genetic outcomes: targeted point mutation, targeted genomic deletion of up to 100 kb and targeted insertion of small genetic elements concomitant with large genomic deletions.

Targeting DNA Double-Strand Breaks with TAL Effector Nucleases

A new class of sequence-specific nucleases created by fusing transcription activator-like effectors (TALEs) to the catalytic domain of the FokI endonuclease is reported.

Genetic engineering of human ES and iPS cells using TALE nucleases

The data suggest that TALENs employing the specific architectures described here mediate site-specific genome modification in human pluripotent cells with similar efficiency and precision as do zinc-finger nucleases (ZFNs).

Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome.

The use of designed zinc finger nucleases (ZFNs) for efficient transgenesis without drug selection into the PPP1R12C gene, a "safe harbor" locus known as AAVS1, allows bona fide isogenic settings for high-throughput functional genomics, proteomics, and regulatory DNA analysis in essentially any transformed human cell type and in primary cells.

Generation of a triple‐gene knockout mammalian cell line using engineered zinc‐finger nucleases

The development of zinc‐finger nucleases (ZFNs) targeted to cleave three independent genes with known null phenotypes are reported, demonstrating the utility of ZFNs in multi‐locus genome engineering.

Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases

A rapid single-step approach to targeted gene knockout in mammalian cells, using engineered zinc-finger nucleases (ZFNs), to establish a new method for gene knockout with application to reverse genetics, functional genomics, drug discovery, and therapeutic recombinant protein production is demonstrated.

In Situ Genetic Correction of the Sickle Cell Anemia Mutation in Human Induced Pluripotent Stem Cells Using Engineered Zinc Finger Nucleases

The generation of iPSC lines from sickle cell anemia patients and in situ correction of the disease causing mutation using three ZFN pairs made by the publicly available oligomerized pool engineering method (OPEN) provide an important proof of principle that ZFNs can be used to produce gene‐corrected human iPSCs that could be used for therapeutic applications.