ke10050  (Proteintech)

Journal: The Innovation

Article Title: mRNA-engineered CRISPR-Cas epigenetic editors enable durable and efficient gene silencing in vivo

doi: 10.1016/j.xinn.2025.101151

Figure Lengend Snippet: Optimization of SpCas9-OFF and SF01-OFF EE mRNA for efficient gene silencing (A) Schematic of V1 (unoptimized) and V2 (optimized) mRNA constructs. The V1 construct represents unoptimized mRNA, with coding sequences (CDSs) amplified from previously described mammalian expression plasmids. The V2 version incorporates codon optimization, modified 5ʹ/3ʹ UTRs, altered linkers, and revised nuclear localization signals (NLSs). (B and C) Comparison of silencing efficiency for V1 and V2 mRNA constructs. Silencing of Snrpn -GFP (in reporter cells) and endogenous genes ( CD29 , CD81 , and CD151 in HEK293T cells) was assessed 21 days post-electroporation. Data are the mean of three biological replicates. p values from two-way ANOVA; ∗∗∗∗ p ≤ 0.0001 . (D) Diagram illustrating the Hepa1-6 Pcsk9 IRES-EGFP reporter cell line, where EGFP expression is coupled to endogenous Pcsk9 transcription. An internal ribosome entry site (IRES)-EGFP cassette was integrated between the terminal exon and the 3′ UTR of the endogenous Pcsk9 gene. (E) Strategy for sgRNA and crRNA screening using the Hepa1-6 Pcsk9 IRES-GFP reporter cell line with SpCas9-OFF-EE or SF01-OFF-EE, respectively. Top dashed box: SF01 crRNA binding sites. Bottom dashed box: SpCas9 sgRNA binding sites. The x axis position of each line indicates the target site relative to the Pcsk9 transcriptional start site (TSS; in nucleotides). Light blue lines: crRNAs targeting the plus strand; red lines: crRNAs targeting the minus strand. Light green shading: two regions targeted by crRNAs that induced robust EGFP silencing with SF01-OFF-EE. Purple lines: previously described sgRNAs (na-sgRNA). Orange lines: 10 sgRNAs designed around the na-sgRNA binding sites. (F and G) Evaluation of EGFP silencing efficacy and durability in Hepa1-6 Pcsk9 IRES-EGFP cells for up to 1 week, using 10 selected sgRNAs (F) and 41 crRNAs (G) from (E). (H and I) Corresponding CpG methylation levels at Pcsk9 promoter for the guide RNAs screened in (F) and (G), analyzed by targeted bisulfite sequencing. Data in (F)–(I) represent the mean ± SD from n = 3 independent biological replicates.

Article Snippet: Serum was separated by centrifugation at 2,000 × g for 120 min. PCSK9 levels were determined by ELISA using the Mouse PCSK9 ELISA Kit (Proteintech, KE10050) following the manufacturer’s instructions.

Techniques: Construct, Amplification, Expressing, Modification, Comparison, Electroporation, Binding Assay, CpG Methylation Assay, Methylation Sequencing

Journal: The Innovation

Article Title: mRNA-engineered CRISPR-Cas epigenetic editors enable durable and efficient gene silencing in vivo

doi: 10.1016/j.xinn.2025.101151

Figure Lengend Snippet: In vitro screening and multi-omics specificity of Pcsk9 -targeting editors in mouse liver cells (A and B) Evaluation of EGFP silencing in reporter cells 21 days after treatment with SpCas9-OFF-EE and multiplexed sgRNAs (A) or SF01-OFF-EE and multiplexed crRNAs (B). Data are the mean ± SD ( n = 3). (C and D) Corresponding quantification of CpG methylation at the Pcsk9 promoter for the treatments in (C) and (D). Data are the mean ± SD ( n = 3). (E) Quantification of secreted PCSK9 protein from Hepa1-6 cells 21 days after treatment with various editors, measured by ELISA and normalized to mock controls. Data represent the mean of triplicate measurements. (F) Bar plot of global CpG methylation levels determined by WGBS, showing no significant global changes across treatments ( n = 3). (G) CpG methylation profiles across the Pcsk9 locus in Hepa1-6 cells treated with different editor constructs, showing targeted hypermethylation at the CGI (yellow rectangle). (H and I) Manhattan plots of genome-wide methylation changes (WGBS) comparing SpCas9-OFF-EE- (H) or SF01-OFF-EE- (I) treated cells to mock controls. Benjamini-Hochberg false discovery rate (FDR)-adjusted p values (DSS Wald test, two-sided) for each CpG are plotted against their genomic coordinates. Differentially methylated CpGs (DMCs) within the Pcsk9 differentially methylated region (DMR) are shown in red. The DMR threshold was set as a p value (DSS Wald test, two-sided, unadjusted) < 1 × 10 −10 . (J) Volcano plots from RNA-seq analyses showing differentially expressed genes (DEGs) from RNA-seq analysis for SpCas9-OFF-EE (left) and SF01-OFF-EE (right) treatments ( n = 3 per experimental condition). p values were from the Wald test for binomial distributions, adjusted for multiple testing using the Benjamini-Hochberg method. The horizontal dashed line indicates the threshold on the adjusted p value (FDR ≤ 0.05), and the vertical dashed lines represent the threshold on |log2 FC| ≥ 1. Upregulated genes are in red, downregulated genes are in light blue, and non-DEGs are in gray. The Pcsk9 gene is indicated. (K and L) Scatterplots correlating methylation changes of DMRs with gene expression changes for SpCas9-OFF-EE (K) and SF01-OFF-EE (L). The analysis highlights that Pcsk9 (blue dot) is the primary target with both significant hypermethylation and transcriptional repression. Off-target DMR-associated gene expression changes are shown in cyan. The Pcsk9 gene is highlighted in blue. Thresholds (gray dashed lines) are set as a methylation (beta value) difference > 0.2 or < −0.2 and RNA-seq log2FC > 1 or < −1.

Article Snippet: Serum was separated by centrifugation at 2,000 × g for 120 min. PCSK9 levels were determined by ELISA using the Mouse PCSK9 ELISA Kit (Proteintech, KE10050) following the manufacturer’s instructions.

Techniques: In Vitro, Biomarker Discovery, CpG Methylation Assay, Enzyme-linked Immunosorbent Assay, Construct, Genome Wide, Methylation, RNA Sequencing, Gene Expression

Journal: The Innovation

Article Title: mRNA-engineered CRISPR-Cas epigenetic editors enable durable and efficient gene silencing in vivo

doi: 10.1016/j.xinn.2025.101151

Figure Lengend Snippet: Efficient in vivo epigenetic silencing of Pcsk9 in mice via LNP-mediated mRNA delivery (A and B) In vivo screen of eight LNP formulations for liver delivery. C57BL/6 mice ( n = 3 per group) received LNPs encapsulating SpCas9 nuclease mRNA and a Pcsk9 -targeting sgRNA. Serum PCSK9 levels (A) and liver indel frequencies (B) were measured at day 7. Dots represent data from individual mice; bars indicate the median for each group. (C and D) Dose-dependent effects of a single injection of LNP8 formulation containing SpCas9-OFF-EE-V2 or SF01-OFF-EE-V2 mRNA and cognate Pcsk9 -targeting sgRNAs or crRNAs. Circulating PCSK9 levels (C) and plasma LDL-C levels (D) in C57BL/6 mice ( n = 6 per group) were measured at day 7 post-injection. (E and F) Comparison of silencing efficacy of PCSK9 levels among different editor systems at a 3.0 mg/kg dose. PCSK9 levels (E) and plasma LDL-C (F) levels were measured at day 7 ( n = 6 per group) post-injection of LNP8 formulated containing various editor payloads (SpCas9 nuclease, SpCas9-OFF-EE-V2, 573-split-SpCas9-OFF-EE, 713-split-SpCas9-OFF-EE, SF01 nuclease, or SF01-OFF-EE-V2) along with cognate Pcsk9 -targeting sgRNAs or crRNAs. Data points represent individual mice; bars indicate the mean ± SD.

Article Snippet: Serum was separated by centrifugation at 2,000 × g for 120 min. PCSK9 levels were determined by ELISA using the Mouse PCSK9 ELISA Kit (Proteintech, KE10050) following the manufacturer’s instructions.

Techniques: In Vivo, Injection, Formulation, Clinical Proteomics, Comparison

Journal: The Innovation

Article Title: mRNA-engineered CRISPR-Cas epigenetic editors enable durable and efficient gene silencing in vivo

doi: 10.1016/j.xinn.2025.101151

Figure Lengend Snippet: Comprehensive in vivo specificity profiling of Pcsk9 silencing in mouse liver (A and C) Manhattan plots from WGBS of liver tissue, comparing mice treated with SpCas9-OFF-EE (A) or SF01-OFF-EE (C) to vehicle controls. The most significant DMR is at the Pcsk9 locus (red box). Benjamini-Hochberg false discovery rate (FDR)-adjusted p values (DSS Wald test, two-sided) for each CpG are plotted against their genomic coordinates. Differentially methylated CpGs (DMCs) within the Pcsk9 differentially methylated region (DMR) are shown in red. The DMR threshold was set as a p value (DSS Wald test, two-sided, adjusted) < 1 × 10 −10 . (B and D) Scatterplots correlating methylation differences (DMRs) with gene expression changes (DEGs) for SpCas9-OFF-EE (B) and SF01-OFF-EE (D). Pcsk9 (blue dot) is the primary concordant off target. Scatterplots were generated to examine the correlation between methylation differences in DMRs (from WGBS, y axis) and the log 2 fold change (FC) in gene expression (from RNA-seq, x axis) for all genes located within ±20 kb of each DMR in liver tissue. The analysis compared the SpCas9-OFF-EE group with the LNP vehicle-only control and SF01-OFF-EE versus LNP vehicle-only control. The Pcsk9 gene is highlighted in blue. Thresholds (gray dashed lines) are set as a methylation (beta value) difference > 0.2 or < −0.2 and RNA-seq log2FC > 1 or < −1. DEG, differentially expressed gene. (E) Global CpG methylation levels in liver tissue were unchanged across treatment groups ( n = 3 per experimental condition). (F) Volcano plots from RNA-seq of liver tissue, showing Pcsk9 as the most significantly downregulated gene in mice treated with SpCas9-OFF-EE (left) and SF01-OFF-EE (right) compared to controls ( n = 6 per group). p values were determined using the Wald test for binomial distributions and adjusted for multiple testing (Benjamini-Hochberg method). The horizontal dashed line indicates the threshold on the adjusted p value (FDR ≤ 0.05), and the vertical dashed lines represent the FC threshold on |log2 FC| ≥ 1. Upregulated genes are in red, downregulated genes are in light blue, and non-DEGs are in gray. The Pcsk9 gene is indicated. (G) Gene Ontology (GO) biological process pathway enrichment analysis of DEGs identified by RNA-seq in livers reveals significant enrichment of cholesterol and steroid metabolic pathways in both treatment groups, consistent with Pcsk9 silencing.

Article Snippet: Serum was separated by centrifugation at 2,000 × g for 120 min. PCSK9 levels were determined by ELISA using the Mouse PCSK9 ELISA Kit (Proteintech, KE10050) following the manufacturer’s instructions.

Techniques: In Vivo, Methylation, Gene Expression, Generated, RNA Sequencing, Control, CpG Methylation Assay

Journal: The Innovation

Article Title: mRNA-engineered CRISPR-Cas epigenetic editors enable durable and efficient gene silencing in vivo

doi: 10.1016/j.xinn.2025.101151

Figure Lengend Snippet: Durable in vivo silencing of Pcsk9 for 180 days (A) Schematic of the long-term in vivo study design. C57BL/6 mice received a single intravenous injection of LNP-formulated editors. Blood and liver tissues were collected at the indicated time points for analysis. (B and C) Time-course analysis of circulating PCSK9 (B) and LDL-C (C) levels for 180 days. A single dose of SpCas9-OFF-EE, SF01-OFF-EE, and split-SpCas9-OFF-EE editors resulted in stable, long-term suppression ( n = 6 mice per group). Data are the mean ± SD. (D and E) WGBS profiles of the Pcsk9 locus in liver tissue at days 30 (D) and 180 (E) post-injection, showing robust and sustained CpG hypermethylation at the promoter CGI (orange rectangle) in editor-treated groups compared to mock controls.

Article Snippet: Serum was separated by centrifugation at 2,000 × g for 120 min. PCSK9 levels were determined by ELISA using the Mouse PCSK9 ELISA Kit (Proteintech, KE10050) following the manufacturer’s instructions.

Techniques: In Vivo, Injection