Chemogenetic System Demonstrates That Cas9 Longevity Impacts Genome Editing Outcomes

Abstract

Prolonged Cas9 activity can hinder genome engineering as it causes off-target effects, genotoxicity, heterogeneous genome-editing outcomes, immunogenicity, and mosaicism in embryonic editing—issues which could be addressed by controlling the longevity of Cas9. Though some temporal controls of Cas9 activity have been developed, only cumbersome systems exist for modifying the lifetime. Here, we have developed a chemogenetic system that brings Cas9 in proximity to a ubiquitin ligase, enabling rapid ubiquitination and degradation of Cas9 by the proteasome. Despite the large size of Cas9, we were able to demonstrate efficient degradation in cells from multiple species. Furthermore, by controlling the Cas9 lifetime, we were able to bias the DNA repair pathways and the genotypic outcome for both templated and nontemplated genome editing. Finally, we were able to dosably control the Cas9 activity and specificity to ameliorate the off-target effects. The ability of this system to change the Cas9 lifetime and, therefore, bias repair pathways and specificity in the desired direction allows precision control of the genome editing outcome.

A Chemical Toolbox for Labeling and Degrading Engineered Cas Proteins

“… 30 The result suggested that the PROTAC-FCPF-degraded Cas9 FCPF efficiently repressed the biological function of the CRISPR-Cas9 FCPF system in a time-controlled fashion. In agreement with the result reported by Sreekanth et al, 24 the incubation of PROTAC-FCPF (1 μM) for 24 h reduced the off-target effect from 12.1% to 3.56% ( Figure S2H ), implying that Cas9 degraders can reduce the off-target effects of a CRISPR/Cas9 system.…”

“…They demonstrated that the small molecular degrader, dTAG-47, degraded Cas9 carrying two FK506-binding proteins (FKBPs, Cas9 FKBP ) at the N-terminal and loop region at concentrations of 0.1–1 μM in a 24 h treatment, which significantly increased the specificity of the CRISPR-Cas9 system. 24 We evaluated the activity of PROTAC-FCPF for Cas9 FCPF degradation in both 8 and 24 h treatments. We found that 10 μM PROTAC-FCPF robustly degraded the Cas9 FCPF protein ( Figure 2 C and Figure S2C ).…”

“…Very recently, Choudhary and his co-workers developed a dTAG-based PROTAC system to degrade Cas9 tagging FKPB. 24 They demonstrated significantly improved activity and specificity of Cas9 FKPB -mediated genome editing under the control of a dTAG molecule. This new development highlights the potential application of Cas9 degraders in CRISPR-Cas9 genome editing.…”

“… 26 , 47 In the case of Cas9 FKBP , two insertions were required for a sufficient degradation. 24 In contrast, an insertion of the FCPF sequence represents a minimal modification of the target protein. Therefore, multiple FCPF sequences could be inserted into a target protein, which makes this technology applicable for an endogenously expressed protein in combination with a CRISPR-Cas9 knockin system.…”

Strategies for Post-Translational Control of Protein Expression and Their Applications

“…Because off-target editing is often slower than on-target editing, regulating the expression of Cas9 improves on-target editing while avoiding off-target editing and subsequent genotoxicity. Several research groups have demonstrated that controlling Cas9 expression levels reduces off-target editing [97][98][99][100][101]. Furthermore, the Cre protein can also be fused with DD to achieve inducible control of gene expression [102].…”