Engineered Cas9 variants bypass Keap1-mediated degradation in human cells and enhance epigenome editing efficiency

Abstract

As a potent and convenient genome-editing tool, Cas9 has been widely used in biomedical research and evaluated in treating human diseases. Numerous engineered variants of Cas9, dCas9 and other related prokaryotic endonucleases have been identified. However, as these bacterial enzymes are not naturally present in mammalian cells, whether and how bacterial Cas9 proteins are recognized and regulated by mammalian hosts remain poorly understood. Here, we identify Keap1 as a mammalian endogenous E3 ligase that targets Cas9/dCas9/Fanzor for ubiquitination and degradation in an 'ETGE'-like degron-dependent manner. Cas9-'ETGE'-like degron mutants evading Keap1 recognition display enhanced gene editing ability in cells. dCas9-'ETGE'-like degron mutants exert extended protein half-life and protein retention on chromatin, leading to improved CRISPRa and CRISPRi efficacy. Moreover, Cas9 binding to Keap1 also impairs Keap1 function by competing with Keap1 substrates or binding partners for Keap1 binding, while engineered Cas9 mutants show less perturbation of Keap1 biology. Thus, our study reveals a mammalian specific Cas9 regulation and provides new Cas9 designs not only with enhanced gene regulatory capacity but also with minimal effects on disrupting endogenous Keap1 signaling.

Graphical Abstract

Figure 1.

Keap1 interacts with SpCas9 in an ‘ETGE’ degron-dependent manner. (A) A cartoon illustration of SpCas9 protein domain structures with the putative Keap1 degron ‘ETGE’ labeled in red. (B) A protein sequence alignment for indicated Cas9 or Cas9-like proteins from indicated species with UniProt numbers. Potential Keap1 ‘ETGE’ or ‘ETGE’-like motifs are indicated. (C, D) Immunoblot (IB) analysis of whole cell lysates (WCL) and Flag-immunoprecipitants (IP) derived from HEK293 cells transfected with HA-Keap1 and/or indicated Flag-SpCas9 constructs for 48 h. (E) A structure simulation by PyMOL indicating that the SpCas9-ETGE motif (blue, PDB: 4oo8) exerts a similar structure topology as NRF2-ETGE motif (red, PDB: 2flu). (F) A representative Coomassie staining image for SDS-PAGE for Flag-IPs derived from HEK293T cells transfected with EV or WT-SpCas9 for 48 h. (G) A volcano plot indicating that multiple E3 ubiquitin ligases were identified from a proteomic analysis using samples from (F) to establish a SpCas9 interactome. (H) A representative tandem mass spectrum indicating identification of the KEAP1 peptide from SpCas9 interactome along with a reference spectrum from the SRMAtlas (80) database. Inset: Correlation between observed retention times (RT) and Chronologer (81) predicted retention times (iRT).

Figure 2.

Keap1 targets SpCas9 for ubiquitination and degradation. (A, B) IB analysis of WCL derived from HEK293T cells transfected with indicated Flag-SpCas9 and indicated doses of HA-Keap1 cDNA (μg) for 48 hrs. (C) IB analysis of WCL derived from control or endogenous Keap1-depleted HEK293 cells by shRNAs transfected with either WT- or EAAE-SpCas9 constructs for 48 h. (D) IB analysis of WCL derived from control or endogenous Keap1-depleted HBE cells by shRNAs transfected with either WT- or EAAE-SpCas9 constructs for 48 h. (E) IB analysis of WCL derived from control or endogenous Keap1-depleted HEK293 cells by shRNAs transfected with indicated doses of WT-SpCas9 construct (μg) for 48 h. (F) IB analysis of WCL derived from control or endogenous Keap1-depleted HEK293 cells by shRNAs transfected with indicated Flag-SpCas9 and HA-Keap1 (resistant to shKeap1) constructs for 48 h. (G) IB analysis of WCL derived from HEK293 cells transfected with either WT- or EAAE-SpCas9 constructs for 48 h. Where indicated, 10 μM MG132 was added to cell culture media overnight before cell collection. (H) IB analysis of Ni-NTA pulldowns and WCL from HEK293 cells transfected with indicated DNA constructs for 48 h. 10 μM MG132 was added to cell culture media overnight before cell collection. (I, J) IB analysis of WCL derived from HEK293 cells transfected with either WT- or EAAE-SpCas9 constructs and treated with indicated compounds. Where indicated, DMSO, CDDO (50 nM) or tBHQ (10 μM) was added to cell culture media for 16 h before cell collection.

Figure 3.

Keap1 binds and targets SaCas9 and Fanzor for degradation. (A, E) The protein sequence alignment for potential ‘ETGE’-like motifs from SaCas9 (A) and Fanzor (E) to SpCas9. (B, D) IB analyses of WCL from HEK293 cells transfected with HA-SaCas9 and indicated amounts of Flag-Keap1 constructs (μg) for 48 h. (C) IB analyses of Flag-IP and WCL from HEK293 cells transfected with indicated HA-SaCas9 constructs with Flag-Keap1 for 48 h. (F) IB analyses of WCL from HEK293T cells transfected with Flag-Fanzor with increasing doses of HA-Keap1 constructs for 48 h. (G) IB analyses of Flag-IPs and WCL from HEK293T cells transfected with HA-Keap1 and indicated Flag-Fanzor constructs for 48 h. (H) IB analyses of WCL from HEK293T cells transfected with indicated Flag-Fanzor constructs with or without HA-Keap1 for 48 h.

Figure 4.

Engineered Keap1-degron mutated SpCas9 or SaCas9 variants display enhanced Cas9-mediated genome editing ability in vitro. (A) An illustration of the sgRNA sequence targeting the human AAVS1 locus used in CRISPR-mediated knockout efficiency tests and the guide RNA sequence used in this study to introduce a KpnI site for knockin efficacy tests. (B) IB analysis of WCL derived from HEK293T cells transfected with Flag-SpCas9 with or without HA-Keap1 constructs for 72 h. (C, E, F, H, Q) Representative DNA agarose gel images of standard T7E1 assays derived from HEK293T cells transfected with indicated Cas9 and sgRNA performed 3-day post-transfection. Indel% was calculated and presented to indicate target gene editing efficiency. (D) IB analysis of WCL derived from control or endogenous Keap1-depleted HEK293T cells transfected with indicated Cas9 constructs and EMX1-sgRNAs. Where indicated, lentiviruses for shscramble or shKeap1-11 were used to infect HEK293 cells for 24 h followed by 72 h selection with 1 μg/ml puromycin to eliminate non-infected cells. (G) IB analysis of WCL derived from HEK293T cells transfected with EV (empty vector), WT- or EAAE-SpCas9-Flag and AAVS1-sgRNAs for 72 h. (I) Left: IB analysis of WCL derived from HEK293T cells transfected with EV (empty vector), WT- or EAAE-SpCas9-Flag and EMX1-sgRNAs for 72 h; Right: representative DNA agarose gel images of standard T7E1 assays derived from HEK293T cells transfected with indicated Cas9 and sgRNA performed 3-day post-transfection. Indel% was calculated and presented to indicate target gene editing efficiency. (J–L) Left: IB analysis of WCL derived from HEK293T (J), HBE (K) or BPH1 (L) cells transfected with EV (empty vector), WT- or EAAE-SpCas9-Flag and EMX1-sgRNAs for 72 h; Right: calculations of EMX-1 gene editing efficiency derived from DNA sanger sequencing of PCR products of targeted EMX-1 genomic regions from HEK293T cells transfected with indicated Cas9 and sgRNA performed 3-day post-transfection. n = 2 (biological duplicates). *P< 0.05 (one-way ANOVA test). (M) The scheme to test CRISPR_ki efficiency by introducing a new Sca-I site in the mouse Rosa26 locus. (N) IB analysis of WCL derived from MEF cells transfected with WT or EAAE-SpCas9-Flag constructs for 48 h. (O) A representative DNA agarose gel image of Sca-I digestion of PCR products using genomic DNA from cells from (N) to measure CRISPR_ki efficiency. (P) IB analysis of WCL derived from HEK293T cells transfected with EV (empty vector), WT- or AEGA-SaCas9-Flag and hDMD-sgRNAs for 72 h. (R) Calculations of hDMD gene editing efficiency derived from DNA sanger sequencing of PCR products of targeted hDMD genomic regions from HEK293T cells transfected with indicated Cas9 and sgRNA performed 3-day post-transfection. n = 2 (biological duplicates). *P< 0.05 (one-way ANOVA test). (S, T) Left: IB analysis of WCL derived from BPH1 cells transfected with EV (empty vector), WT- or AEGA-SaCas9-HA and indicated sgRNAs for 48 h; Right: calculations of indicated gene editing efficiency derived from DNA sanger sequencing of PCR products of targeted genomic regions from BPH1 cells transfected with indicated Cas9 and sgRNA performed 3-day post-transfection. n = 2 (biological duplicates). *P< 0.05 (one-way ANOVA test).

Figure 5.

dCas9-EAAE-fusions exert enhanced epigenome regulation ability in cells. (A) A cartoon illustration of both dCas9-WT-p300 fusion and dCas9-EAAE-p300 fusion proteins in bringing the p300 transcription activator to a given site determined by sgRNA in regulating expression of genes of interests. (B) IB analyses of cytoplasm or chromatin fractions from HEK293T cells transfected with WT- or EAAE-dCas9-p300 constructs for 48 h. (C) Fluorescent reporter assays derived from HEK293T cells transfected with WT-dCas9-p300 or EAAE-dCas9-p300 constructs with a fluorescence reporter indicating that dSpCas9-EAAE-p300 fusion proteins displays an increase in activating targeted exogenous gene expression compared with dSpCas9-WT-p300. Fluorescent signals were detected by FACS analyses against GFP. n = 3 (biological triplicates). *P< 0.05 (one-way ANOVA test). (D) RT-PCR analyses of expression of indicated mRNA targets from HEK293T cells transfected with WT-dCas9-p300 or EAAE-dCas9-p300 with sgRNAs targeting either OCT4 or IL1RN, which indicates that dSpCas9-EAAE-p300 fusion proteins displays a significantly increased efficiency in activating targeted endogenous gene expression compared with dSpCas9-WT-p300. n = 3 (biological triplicates). (E) IB analysis of chromatin fractions of HEK293T cells transfected with indicated WT- or EAAE-HA-dCas9-p300 constructs for 48 h. Where indicated, 200 μg/ml CHX was added to culture and cells were harvested at indicated time periods. Quantifications of HA-dCas9-p300 protein half-life were presented in (F). (G) IB analyses cytoplasm and chromatin fractions from HEK293T cells transfected with either WT- or EAAE-dCas9-CRAB for 48 h. (H) IB analysis of chromatin fractions of HEK293T cells transfected with indicated WT- or EAAE-HA-dCas9-CRAB constructs for 48 h. Where indicated, 200 μg/ml CHX was added to culture and cells were harvested at indicated time periods. Quantifications of HA-dCas9-CRAB protein half-life were presented in (I). (J–O) Lower panels: IB analyses of WCL from HEK293T cells transfected with indicated dCas9-CRAB and sgRNAs. Upper panels: RT-PCR analyses of mRNAs from cells in lower panels expressing either WT- or EAAE-dCas9-CRAB for indicated target gene expression. Notably, U6 snRNA is used as an internal normalization control. n = 3 (biological triplicates). *P< 0.05 (one-way ANOVA test).

Figure 6.

SpCas9 stabilizes endogenous Keap1 substrates by competitively binding Keap1 to modulate its cellular functions. (A) IB analysis of WCL derived from HEK293 cells transfected with Flag-NRF2 and increasing doses of Flag-SpCas9 constructs for 48 h. (B) IB analysis of WCL derived from HEK293 cells transfected with Flag-NRF2 and indicated WT-Cas9 or EAAE-Cas9 constructs for 48 h. (C) IB analysis of WCL and HA-IPs derived from HEK293 cells transfected with HA-Keap1, Flag-NRF2 or Flag-SpCas9 constructs for 48 h. (D) IB analysis of WCL derived from HEK293 cells transfected with HA-SpCas9 with increasing doses of Flag-NRF2 constructs for 48 h. (E) IB analysis of WCL and Flag-IPs derived from HEK293 cells transfected with Flag-PALB2, HA-Keap1 and increasing doses of HA-SpCas9 constructs for 48 h. (F) IB analysis of WCL derived from HEK293 cells transfected with Flag-PALB2 and increasing doses of HA-SpCas9 constructs for 48 h. (G) IB analysis of WCL derived from HEK293 cells transfected with HA-SpCas9 and increasing doses of Flag-PALB2 constructs for 48 h. (H) A proposed model for how SpCas9 expression modulates cellular function. Specifically, SpCas9 binds and titrates Keap1 away from endogenous Keap1 substrates, leading to stabilization of Keap1 substrates including NRF2 and subsequently altered cellular signaling. (I) IB analysis of WCL derived from HEK293 cells transfected with Flag-NRF2 with indicated HA-Keap1 constructs for 48 h. (J) IB analysis of WCL derived from HEK293 cells transfected with Flag-SpCas9 with indicated HA-Keap1 constructs for 48 h. (K) A schematic illustration of cancer patient derived Kelch domain mutations in Keap1. (L) IB analysis of WCL from HEK293T cells transfected with Flag-NRF2 with indicated HA-Keap1 constructs. (M) IB analysis of WCL from HEK293T cells transfected with Flag-SpCas9 with indicated HA-Keap1 constructs. (N) A table summarizing various Keap1 mutants used in this study with impaired function in either degrading NRF2 or Cas9. (O) IB analysis of WCL from HEK293T cells transfected with Flag-SpCas9 with indicated HA-Keap1 constructs and EMX1-sgRNA for 72 h. (P) A representative DNA agarose gel image of the standard T7E1 assays using cells from (O).