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.

Keywords: CRISPR; Cas nuclease; genome editing; guide RNA; small molecule; specificity.

Figure 1

(A) Cas-nuclease-induced double-strand break (DSB) is repaired by endogenous cellular pathways. (B) Cas nucleases often tolerate partial mismatches and induce off-target DNA cleavages.

Figure 2

Examples of the small-molecule control of Cas nuclease expression. (A) Transcriptional control of Cas nuclease expression by doxycycline. Temporal or spatiotemporal control of the nuclease expression can be achieved by employing appropriate promoters for rtTA expression. (B) Small-molecule control of Cas9 translation using stop-codon read-through strategies. (C) Small-molecule control of mRNA export to modulate the expression of genome-editing machinery.

Figure 3

Discovery of anti-CRISPR molecules to inhibit native Cas nucleases. (A,B) Anti-CRISPR molecules display different modes of action, including (A) inhibition of the Cas nuclease−DNA interactions, and (B) inhibition of the nuclease domains. (C) A high-throughput in vitro fluorescence polarization assay to detect SpCas9−PAM (protospacer adjacent motif) interactions. (D) An image-based high-content assay to identify SpCas9 inhibitors in human cells. (E) A cell-based assay to identify SpCas9 inhibitors in E. coli. (F) A high-throughput in vitro FRET assay to identify SpCas9 inhibitors.

Figure 4

Examples of small-molecule control of engineered Cas nucleases. (A) Targeted degradation of SpCas9-FKBP^F12V fusion by dTAG-47 molecule to switch off genome editing. (B) Destabilized SpCas9-DHFR fusion is stabilized by TMP to switch on genome editing. (C) SpCas9 fused to an engineered intein is spliced by binding to 4HT, and the active SpCas9 is released. (D) Split SpCas9 is dimerized by rapamycin to reconstitute active SpCas9. (E) SpCas9-ERT2 fusion in the cytoplasm is translocated to the nucleus upon binding of 4HT to the ERT domain. (F) SpCas9 activity is blocked by the autoinhibitory BCL-xL−BH3 interaction but restored by the inhibitors of the protein−peptide interaction.

Figure 5

Examples of small-molecule control of engineered gRNAs. (A) gRNAs are fused with an aptamer that is unstructured, but are stabilized and folded into a functional form by binding to theophylline. (B) gRNAs are fused with an aptazyme that is activated by binding to theophylline and induces the RNA self-cleavage to release functional gRNAs. (C) Mutant gRNAs containing G-G mismatches are recognized and inactivated by NCD. (D) A gRNA masked with AMN groups becomes activated by reacting with phosphines. (E) Active gRNAs that contain small chemical modifications become inactive by the binding or reaction with larger moieties.

Figure 6

Examples of the small-molecule control of engineered Acr proteins. (A) Destabilized AcrIIA4-DHFR fusion is stabilized by TMP to switch off genome editing. (B) Acr proteins fused to an engineered intein are spliced by binding to 4HT, and the activated Acr proteins inhibit genome editing. (C) AcrIIA4 fused to mAID is degraded by auxin that acts as a molecular glue in plant cells, and dSpCas9 is activated.