A Molecular Glue Approach to Control the Half-Life of CRISPR-Based Technologies

Abstract

Cas9 is a programmable nuclease that has furnished transformative technologies, including base editors and transcription modulators (e.g., CRISPRi/a), but several applications of these technologies, including therapeutics, mandatorily require precision control of their half-life. For example, such control can help avert any potential immunological and adverse events in clinical trials. Current genome editing technologies to control the half-life of Cas9 are slow, have lower activity, involve fusion of large response elements (>230 amino acids), utilize expensive controllers with poor pharmacological attributes, and cannot be implemented in vivo on several CRISPR-based technologies. We report a general platform for half-life control using the molecular glue, pomalidomide, that binds to a ubiquitin ligase complex and a response-element bearing CRISPR-based technology, thereby causing the latter's rapid ubiquitination and degradation. Using pomalidomide, we were able to control the half-life of large CRISPR-based technologies (e.g., base editors and CRISPRi) and small anti-CRISPRs that inhibit such technologies, allowing us to build the first examples of on-switch for base editors. The ability to switch on, fine-tune, and switch-off CRISPR-based technologies with pomalidomide allowed complete control over their activity, specificity, and genome editing outcome. Importantly, the miniature size of the response element and favorable pharmacological attributes of the drug pomalidomide allowed control of activity of base editor in vivo using AAV as the delivery vehicle. These studies provide methods and reagents to precisely control the dosage and half-life of CRISPR-based technologies, propelling their therapeutic development.

Figure 1.

Demonstration of Cas9 degradation using superdegron derived from the short, 60-amino-acid pomalidomide-binding domain ZFP91-IKZF3. (A) Schematic showing the proteasomal degradation of Cas9 using the chimeric degron ZFP91-IKZF3 (superdegron) and pomalidomide. (B) Pomalidomide-induced, dose-dependent degradation of transiently nucleofected superdegron-Cas9 constructs was evaluated in U2OS.eGFP.PEST cells using an eGFP disruption assay. After nucleofection, cells are incubated with indicated dose of pomalidomide for 24 h. (C) Schematic showing the NanoBRET-based ternary complex formation between Halotag-cereblon (HT-CRBN), LgBiT reconstituted N-HiBiT-LSD-Cas9 and pomalidomide. (D) milliBRET ratio for the pomalidomide dose-induced ternary complex formation between LSD-Cas9 and HT-CRBN in N-HiBiT-LSD-Cas9 stably expressing U2OS cells. (E, F) Capillary electrophoretic immunoblots showing the pomalidomide-induced dose-dependent (E) and time-dependent (F) degradation of LSD-Cas9 in stably expressing U2OS cells. For the dose–response experiment, cells were incubated with pomalidomide for 12 h (1E); for the time-course experiment, 1 μM pomalidomide was used (1F). (G) Volcano plot depicting the label-free global proteomic analysis of U2OS cells (stably expressing LSD-Cas9) treated with 1 μM pomalidomide or the DMSO control.

Figure 2.

Generalizability of superdegron tags to base editors and CRISPRa/i systems. (A) Schematic of adenine base editor (ABE8e) fused with a single superdegron tag at loop-231 of the Cas9 nickase (ABE8e-SD6). (B) Pomalidomide dose-induced base editor degradation in HEK293T cells transiently transfected with ABE8e and ABE8e-SD6 constructs. After 72 h of transfection and pomalidomide treatment, genomic DNA extracted was analyzed by NGS for the conversion of A • T to G • C. (C) Capillary electrophoretic immunoblot showing pomalidomide-induced degradation of ABE8e-SD6 in HEK293T stable cells. (D) Schematic of the LSD-dCas9-BFP-KRAB system. (E) Functional analysis of LSD-dCas9-BFP-KRAB upon pomalidomide-dependent degradation was carried out in iPSC cells by measuring the transferrin receptor (TFRC) protein levels via FACS. Briefly, iPSCs stably expressing the LSD-dCas9-BFP-KRAB construct were transfected with TFRC-targeting gRNA in the presence and absence of pomalidomide followed by measuring the transferrin receptor levels using FACS by probing them with antibody (F) Pomalidomide dose-induced dCas9 degradation in LSD-dCas9-BFP-KRAB stably expressing human iPSCs were monitored by Capillary electrophoretic immunoblotting. (G) Schematic of LSD-dCas9-VPR system. (H) HEK293T stable cells encoded with a minimally expressed nanoluciferase (NLuc) gene under the cAMP-response Element (CRE) promoter were nucleofected with LSD-dCas9-VPR and gRNA-targeting CRE results in the expression of the luciferase gene. The addition of pomalidomide resulted in a dose-dependent decrease in the luminescence levels as indicative of degradation of the LSD-dCas9-VPR construct. (I) Capillary electrophoretic immunoblot showing the pomalidomide-induced dose-dependent degradation of LSD-dCas9-VPR in stably expressing HEK293T cells.

Figure 3.

Demonstration of AcrIIA4 degradation-mediated switch-on system for Cas9, base editor activation. (A) Schematic showing the proteasomal degradation of AcrIIA4 using the chimeric degron ZFP91-IKZF3 (superdegron) and pomalidomide leads to activation of Cas9. (B) The Cas9-P2A-CSD-AcrIIA4 fusion was investigated for pomalidomide-induced degradation using the eGFP disruption assay. (C, D) Immunoblots for pomalidomide-induced dose-dependent (C) and time-dependent (D) degradation of CSD-AcrIIA4 in HEK293FT cells that stably express 3x-FLG-CSD-AcrIIA4. (E, F) Pomalidomide dose-dependent degradation of CSD-AcrIIA4 activates the adenine base editor (ABE8e) (E) and the cytosine base editor (CBEmax) (F) were measured by % conversion of A.T to G.C and % conversion of C.G to T.A base pair, respectively, by NGS.

Figure 4.

Cas9 half-life can impact DNA repair outcome. (A) U2OS cell line stably expressing the Reduced Library of 48 target sites used to test editing repair outcomes was transfected with the LSD-Cas9 plasmid and treated with 1 μM pomalidomide at different time points after transfection (0–48 h). The genomic DNA was extracted at 120 h post-transfection, and HTS sequencing was performed to analyze the +1 bp insertions, MH deletions, and non-MH deletions (p-values are computed by unpaired t test with Welch’s correction). (B) ddPCR quantification of single-nucleotide exchange at the RBM20 locus in HEK293T cells following templated DNA repair. For this, the LSD-Cas9 plasmid, RBM20 gRNA plasmid, and ssODN template were transfected in HEK293T cells and were treated with pomalidomide at different time points after transfection. Cells were harvested at 72 h post-transfection, and percentages of HDR and NHEJ in the genomic DNA were analyzed by ddPCR analysis. (C) Luminescence-based quantification of HiBiT knock-in at the GAPDH locus in HEK293T cells following templated DNA repair. LSD-Cas9 plasmid, GAPDH gRNA plasmid, and ssODN template were transfected in HEK293T cells and were treated with pomalidomide at different time points after transfection. Cells were lysed 72 h post-transfection and complemented with LgBiT protein to measure the luminescence.

Figure 5.

Timely degradation of CRISPR-associated proteins improves the targeting specificity. (A–D) Impact of Cas9 half-life on targeting specificity was investigated in HEK293T cells. Pomalidomide dose-dependent control (A, C) of on- versus off-target activity of LSD-Cas9 targeting EMX1 (A) and VEGFA (C). Pomalidomide-induced half-life-dependent (B, D) control of on- versus off-target activity of LSD-Cas9 targeting EMX1 (B) and VEGFA (D) (p-values are computed by unpaired t test with Welch’s correction). (E) Time-dependent control on the transcriptome-wide mutations induced by the ABE8e and ABE8e-SD6 upon addition of pomalidomide.

Figure 6.

Superdegron applicability and degradation demonstration of genome editing proteins in mouse models. (A) Intein reconstitution strategy uses two fragments of protein fused to halves of a split intein that splice to reconstitute a full-length protein following coexpression in host cells. (B) Schematic showing the genomic region of Pcsk9 targeted by the gRNA. (C) Schematic showing retro-orbital injection of 5 × 10¹¹ vg of AAVs consisting of split ABE-SD6 in humanized CRBN knock-in C57Bl6/J mice. AAV-injected mice were given 1 week for genome editing before pomalidomide or the vehicle control were administered orally for 2 weeks. After 3 weeks of AAV injection, mice were euthanized, and their liver and blood were harvested to analyze the base editing levels in the liver and the Pcks9 levels in plasma. (D, E) NGS-based analysis showing (D) the conversion of A • T to G • C in the livers and (E) Pcks9 levels in the blood plasma of control and pomalidomide-treated mice. (p-values are computed by unpaired t test with Welch’s correction).