Small Molecules for Enhancing the Precision and Safety of Genome Editing

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome-editing technologies have revolutionized biology, biotechnology, and medicine, and have spurred the development of new therapeutic modalities. However, there remain several barriers to the safe use of CRISPR technologies, such as unintended off-target DNA cleavages. Small molecules are important resources to solve these problems, given their facile delivery and fast action to enable temporal control of the CRISPR systems. Here, we provide a comprehensive overview of small molecules that can precisely modulate CRISPR-associated (Cas) nucleases and guide RNAs (gRNAs). We also discuss the small-molecule control of emerging genome editors (e.g., base editors) and anti-CRISPR proteins. These molecules could be used for the precise investigation of biological systems and the development of safer therapeutic modalities.

Identification of a Cryptic Binding Site in CRISPR-Cas9 for Targeted Inhibition

“…Precision control of Cas9 over the dimensions of dose and time is critical for its therapeutic applications. − An ideal genome editing with Cas9 is to make the intended edits exclusively at specific loci. In the meanwhile, the function of Cas9 must be timely terminated after its job is done, as persistent action can lead to off-target effects.…”

“…In the meanwhile, the function of Cas9 must be timely terminated after its job is done, as persistent action can lead to off-target effects. Other detrimental consequences, such as chromosomal translocations and genotoxicity, are also observed at elevated levels and prolonged activity of Cas9. , …”

“…There are only a few copies of Cas nuclease substrates present in cells, and the nucleases exhibit fast kinetics at on-target sites than at off-target sites. , Thus, precise and safe genome editing can be achieved by restricting the activity or concentrations of gene-editing machinery in a narrow temporal window. To this end, various strategies have been developed, e.g., fusion of Cas9 or its guide RNA to ligand-responsive protein domains or aptamers. ,, However, these approaches diminish the basal nuclease activity, and some pose an increased delivery difficulty. The naturally occurring anti-CRISPR (Acr) proteins can be employed to switch off genome editing, but their proteinaceous nature hampers their timely delivery into cells. − …”

“…To this end, various strategies have been developed, e.g., fusion of Cas9 or its guide RNA to ligand-responsive protein domains or aptamers. ,, However, these approaches diminish the basal nuclease activity, and some pose an increased delivery difficulty. The naturally occurring anti-CRISPR (Acr) proteins can be employed to switch off genome editing, but their proteinaceous nature hampers their timely delivery into cells. − …”

“…Other detrimental consequences, such as chromosomal translocations and genotoxicity, are also observed at elevated levels and prolonged activity of Cas9. 8,9 There are only a few copies of Cas nuclease substrates present in cells, and the nucleases exhibit fast kinetics at ontarget sites than at off-target sites. 10,11 Thus, precise and safe genome editing can be achieved by restricting the activity or concentrations of gene-editing machinery in a narrow temporal window.…”