Understanding off-target effects through hybridization kinetics and thermodynamics

Abstract

In modern biotechnological and medical research, RNA-guided nucleases (RGNs) continue to be highly effective in targeted modification of genomes and the manipulation of gene expression (Sander and Joung 2014; Wang and Wang 2017). In RNA interference (RNAi) and CRISPR (clustered regularly interspaced short palindromic repeats) -Cas (CRISPR-associated protein) systems, RGNs regulate or modify genes through sequence-specific base-pairing between a short interference or single guide RNAs (siRNAs or sgRNAs) and DNA/RNA targets (Bisaria et al. 2017; Shabalina and Koonin 2008). The patterns of base-pairing interactions may modulate RGN binding affinity and reduce off-targeting (Bisaria et al. 2017; Shabalina et al. 2006). 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 CRISPRCas variants, this editorial primarily focuses on offtargeting in CRISPR-Cas systems and comparison with RNAi. CRISPR-Cas proteins are non-specific endonucleases that bind a protospacer adjacent motif (PAM) located in the proximity of the genomic target (Bollen et al. 2018). sgRNA enables the recognition of the target region of interest through complementary base pairing and directs the Cas nuclease there for specific editing. 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). Preliminary screening of potential candidates and prediction of off-target activities were conducted using not only computational and theoretical approaches but also experimental off-target validation (Zhang et al. 2019; Wienert et al. 2019). 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 CassgRNA delivery modifications. Driving improvements to these parameters is a crucial enabler for RGN-based technologies and the realization of its currently Cell Biol Toxicol (2020) 36:11–15 https://doi.org/10.1007/s10565-019-09505-4