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

Abstract

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.

Prediction-based highly sensitive CRISPR off-target validation using target-specific DNA enrichment

“…To date, various methods, mostly based on next-generation sequencing (NGS), have been developed to detect the off-target mutations in vivo 13 . These methods detect genome-wide off-target mutations in an unbiased fashion both inside and outside the cell.…”

Efficient Homology-directed Repair with Circular ssDNA Donors

“…RNA-guided Cas9 1–3 and Cas12a proteins 4, 5 have provided a facile tool for introducing targeted breaks within genomes. These double-strand breaks (DSBs) can be harnessed to engineer the genome through endogenous DNA repair pathways.…”

Understanding off-target effects through hybridization kinetics and thermodynamics

“…Improvement in specificity, on-target cleavage activity, and reduction of off-targetcleavage can be achieved through changes in CRISPRderived nuclease, engineering of sgRNA, and/or Cas-sgRNA delivery modifications. Driving improvements to these parameters is a crucial enabler for RGN-based technologies and the realization of its currently untapped potential (Kim et al 2019;Bisaria et al 2017). We will also discuss the importance of thermodynamic and kinetic properties for RGN specificity estimation and reducing off-targeting through optimization of sgRNAs.…”

“…RGNs exhibit off-target behavior when interactions and modifications are made not only in the intended location (on-target) but also elsewhere in the genome where sequences are similar to the intended target (off-target) (Klein et al 2018;Kempton and Qi, 2019). Nucleotide sequence preferences that improve sgRNA efficiency are substantially different for variable CRISPR-based systems (Kim et al, 2019;Slaymaker et al 2016;Xu et al 2015), which is adapted from diverse bacterial defense systems (Koonin et al 2017;Makarova et al 2006). Thus, due in part to growing interest in CRISPR-Cas variants, this editorial primarily focuses on offtargeting in CRISPR-Cas systems and comparison with RNAi.…”

“…The sgRNA contains a "seed" region, which is especially responsive to mismatches in duplexes with PAMproximal nucleotides, but variants or mutations of the target distal to the PAM also modulate off rates (Boyle et al 2017). The off-target behavior is not surprising due to the divergence of sgRNA targeting systems where different selective pressures result in optimizations of specificities and other important features, such as turnover rates (Kim et al 2019).…”