Evaluating and Enhancing Target Specificity of Gene-Editing Nucleases and Deaminases

Mar 19, 2019Annual review of biochemistry

Improving How Precisely Gene-Editing Tools Target Specific Genes

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Abstract

Programmable nucleases and deaminases may cause off-target activity, leading to potential genomic damage.

These gene-editing tools can modify genomes but may also recognize similar DNA sequences, causing unintended changes.
Off-target activity can result in local mutations or larger genomic rearrangements.
Measuring and limiting genome-wide off-target effects is crucial for safe therapeutic applications.
Various methods exist for identifying off-target sites in both cells and cell-free environments.
Strategies have been developed to reduce off-target activity in programmable gene-editing systems.

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Programmable nucleases and deaminases, which include zinc-finger nucleases, transcription activator-like effector nucleases, CRISPR RNA-guided nucleases, and RNA-guided base editors, are now widely employed for the targeted modification of genomes in cells and organisms. These gene-editing tools hold tremendous promise for therapeutic applications. Importantly, these nucleases and deaminases may display off-target activity through the recognition of near-cognate DNA sequences to their target sites, resulting in collateral damage to the genome in the form of local mutagenesis or genomic rearrangements. For therapeutic genome-editing applications with these classes of programmable enzymes, it is essential to measure and limit genome-wide off-target activity. Herein, we discuss the key determinants of off-target activity for these systems. We describe various cell-based and cell-free methods for identifying genome-wide off-target sites and diverse strategies that have been developed for reducing the off-target activity of programmable gene-editing enzymes.

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Evaluating and Enhancing Target Specificity of Gene-Editing Nucleases and Deaminases

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