// ThermoCas9 — methylation-sensitive Cas9 from Geobacillus thermodenitrificans T12 // Based on: Roth, Shu, Zhao, Trasanidou, Hoffman et al. // "Molecular basis for methylation-sensitive editing by Cas9" // Nature (2026) — DOI: 10.1038/s41586-026-10384-z (open access, CC BY-NC-ND 4.0) // Numerical efficiencies are illustrative ranges drawn from the paper's figures. const CELL_LINES = { MCF7: { name: "MCF-7", long: "Luminal/ER+ breast adenocarcinoma", type: "Cancer", tissue: "Breast" }, MCF10A: { name: "MCF-10A", long: "Non-tumorigenic mammary epithelium", type: "Normal", tissue: "Breast" }, HCT116: { name: "HCT116", long: "Colorectal adenocarcinoma", type: "Cancer", tissue: "Colon" }, HEK293T: { name: "HEK293T", long: "Human embryonic kidney (reference)", type: "Reference", tissue: "Kidney" }, }; // Cell-line pair scenarios mirroring the paper's experimental design. const SCENARIOS = { BREAST: { name: "Luminal Breast Cancer", short: "ER+ BRCA", cancer_line: "MCF7", normal_line: "MCF10A", blurb: "Luminal/ER+ breast cancers exhibit consistent hypomethylation at ESR1 and GATA3 — both luminal-signature transcription factors. ThermoCas9 selectively edits the hypomethylated cancer alleles while the methylated normal alleles remain inaccessible (Roth et al., 2026, Fig. 5).", targets: [ { gene: "GATA3", site: "T18", coords: "Chr 10:8,045,463", pam: "NNNNCGA", beta_cancer: 0.02, beta_normal: 0.31, edit_cancer: 0.78, edit_normal: 0.0, role: "Oncogenic luminal TF", note: "Truncating GATA3 mutations cause dominant-negative loss of luminal differentiation in ~50% of luminal/ER+ tumors. CE-ThermoCas9 RNP achieved up to 78% editing in MCF-7 (Fig. 5e).", }, { gene: "ESR1", site: "T17", coords: "Chr 6:151,690,043", pam: "NNNNCGA", beta_cancer: 0.07, beta_normal: 0.94, edit_cancer: 0.13, edit_normal: 0.0, role: "Oestrogen receptor 1", note: "Sharp methylation differential (β 0.07 vs 0.94) makes ESR1 a textbook example of methylation-locked targeting. Ligand-independent ESR1 mutations drive endocrine-therapy resistance.", }, { gene: "EGFLAM", site: "T11", coords: "Chr 5:38,258,782", pam: "NNNNCGA", beta_cancer: 0.01, beta_normal: 0.49, edit_cancer: 0.10, edit_normal: 0.0, role: "Control locus", note: "Intermediate methylation in MCF-10A (β=0.49) confirms the dose-response: even partial methylation suppresses editing.", }, ], }, COLORECTAL: { name: "Colorectal Carcinoma · proof-of-concept", short: "CRC PoC", cancer_line: "HCT116", normal_line: "HEK293T", blurb: "EMX1 is differentially methylated between HCT116 (cancer-derived, hypermethylated PAM) and HEK293T (reference, unmethylated PAM). ThermoCas9 cleanly edits EMX1 in HEK293T but yields null editing in HCT116 — a binary on/off demonstration of methylation gating (Fig. 5a).", targets: [ { gene: "EMX1", site: "T4", coords: "EMX1 locus, target 4", pam: "NNNNCGA", beta_cancer: 0.95, beta_normal: 0.00, edit_cancer: 0.00, edit_normal: 0.16, role: "CRISPR validation locus", note: "Reverses the cancer/normal pattern: in this pair the 'cancer' line carries the methylation. Demonstrates the on/off behavior cleanly.", }, { gene: "VEGFA", site: "T9", coords: "VEGFA locus, target 9", pam: "NNNNCGA", beta_cancer: 0.00, beta_normal: 0.00, edit_cancer: 0.16, edit_normal: 0.33, role: "Positive control", note: "Unmethylated in both lines → both edit (33% in HEK293T, 16% in HCT116). Confirms the enzyme works when not gated by methylation.", }, { gene: "VEGFA", site: "T3", coords: "VEGFA locus, target 3", pam: "NNNNCGA", beta_cancer: 0.95, beta_normal: 0.95, edit_cancer: 0.00, edit_normal: 0.00, role: "Negative control", note: "Methylated in both lines → null editing in both. Methylation, not chromatin or expression, is the gating step.", }, { gene: "PRDX4", site: "T5", coords: "PRDX4 locus, target 5", pam: "NNNNCGA", beta_cancer: 0.95, beta_normal: 0.00, edit_cancer: 0.00, edit_normal: 0.18, role: "Differentially methylated", note: "Hypermethylated in HCT116, unmethylated in HEK293T. Editing tracks methylation, not cell-line identity.", }, ], }, }; // ThermoCas9 enzyme variants const VARIANTS = { WT: { name: "Wild-type ThermoCas9", abbr: "WT", desc: "Native Type II-C Cas9 from Geobacillus thermodenitrificans T12 (1082 aa). Recognizes 5'-NNNNCGA-3' or 5'-NNNNCCA-3' PAM. Cleavage blocked when the PAM cytosine is 5mC.", activity_scale: 0.55, methylation_sensitive: true, }, CE: { name: "Catalytically Enhanced ThermoCas9", abbr: "CE", desc: "Engineered double mutant Glu655Gly + Asn696Ile from directed evolution of the HNH–RuvC linker (Supplementary Fig. 18). Higher cleavage rate, methylation sensitivity preserved.", activity_scale: 1.0, methylation_sensitive: true, }, D1017A: { name: "ThermoCas9-D1017A", abbr: "D1017A", desc: "Asp1017→Ala. Asp1017 contacts C5* of the PAM through its major groove. Mutation virtually abolishes cleavage activity (Fig. 4c).", activity_scale: 0.05, methylation_sensitive: true, }, S1019A: { name: "ThermoCas9-S1019A", abbr: "S1019A", desc: "Ser1019→Ala. Loses methylation discrimination but retains cleavage activity — separates PAM recognition from methylation sensing (Fig. 4c).", activity_scale: 0.50, methylation_sensitive: false, }, }; // Delivery formats validated in the paper const DELIVERY = { RNP: { name: "RNP nucleofection", short: "RNP", desc: "Pre-assembled ThermoCas9-sgRNA ribonucleoprotein delivered by Lonza nucleofector. Highest efficiency observed: 78% at GATA3 in MCF-7.", efficiency: 1.0, }, mRNA: { name: "ThermoCas9-mRNA + sgRNA", short: "mRNA", desc: "Codon-optimized ThermoCas9-NLS mRNA + chemically modified sgRNA via Lipofectamine. LNP-compatible for in vivo work.", efficiency: 0.7, }, PLASMID: { name: "Plasmid (DNA)", short: "Plasmid", desc: "Co-transfection of ThermoCas9 + sgRNA + EGFP plasmids; FACS-sortable. Reference / lowest efficiency.", efficiency: 0.45, }, }; // Comparison nucleases const NUCLEASES = { thermocas9: { name: "ThermoCas9 (WT)", short: "TC9", color: "var(--accent)", organism: "G. thermodenitrificans T12" }, ce_thermocas9: { name: "Catalytically Enhanced ThermoCas9", short: "CE-TC9", color: "var(--good)", organism: "Engineered (Roth 2026)" }, spcas9: { name: "SpCas9 (S. pyogenes)", short: "SpCas9", color: "var(--fg-dim)", organism: "S. pyogenes", methylation_insensitive: true }, acecas9: { name: "AceCas9 (Acidothermus cellulolyticus)", short: "AceCas9", color: "var(--purple)", organism: "A. cellulolyticus", inhibition_step: "activation" }, }; // Activity vs PAM-cytosine methylation (β value 0–1). // Calibrated to Roth et al. Fig. 1c (target-strand 5mCpG curve) and Fig. 5a/e indel rates. const activityVsMethylation = (nuclease, beta) => { const b = Math.max(0, Math.min(1, beta)); switch (nuclease) { case "spcas9": return 0.85; // methylation-insensitive case "thermocas9": return 0.85 * Math.pow(1 - b, 3.5) + 0.02; // strong inhibition at binding step case "ce_thermocas9": return 0.95 * Math.pow(1 - b, 3.0) + 0.03; // higher peak, similar profile case "acecas9": return 0.80 * Math.pow(1 - b, 2.2) + 0.05; // sensitive but inhibited at activation step default: return 0; } }; const PAM_OPTIONS = ["NNNNCGA", "NNNNCCA"]; const TC9_FACTS = { organism: "Geobacillus thermodenitrificans T12", type: "Type II-C Cas9", size_aa: 1082, optimal_temp_C: [37, 55], pam_options: PAM_OPTIONS, pam_critical_residue: "Asp1017", methylation_sensing_residue: "Ser1019", activation_residues: ["Lys711", "Asp723"], hnh_rotation_deg: 180, pdb_pre_cleavage: "9AR4", pdb_post_cleavage: "9AR7", resolution_pre_A: 2.8, resolution_post_A: 2.2, paper: { title: "Molecular basis for methylation-sensitive editing by Cas9", authors: "Roth MO, Shu Y, Zhao Y, Trasanidou D, Hoffman RD, et al.", journal: "Nature", year: 2026, doi: "10.1038/s41586-026-10384-z", url: "https://www.nature.com/articles/s41586-026-10384-z", doi_url: "https://doi.org/10.1038/s41586-026-10384-z", }, }; const DEFAULT_TARGET_INDEX = { BREAST: 0, COLORECTAL: 0 }; function hashStr(s) { let h = 2166136261; for (let i = 0; i < s.length; i++) { h ^= s.charCodeAt(i); h = Math.imul(h, 16777619); } return h >>> 0; } function mulberry32(a) { return () => { a = (a + 0x6D2B79F5) >>> 0; let t = a; t = Math.imul(t ^ (t >>> 15), t | 1); t ^= t + Math.imul(t ^ (t >>> 7), t | 61); return ((t ^ (t >>> 14)) >>> 0) / 4294967296; }; } Object.assign(window, { CELL_LINES, SCENARIOS, VARIANTS, DELIVERY, NUCLEASES, activityVsMethylation, PAM_OPTIONS, TC9_FACTS, DEFAULT_TARGET_INDEX, hashStr, mulberry32, });