Can you help me with making a presentation for seminar on CRISPR cas 9 in biochemistry 1st year mbbs
Asking for Preferences
Reading File
Reading File
Reading File
Finding Sources
Searching PubMed
CRISPR Cas9 clinical trials gene therapy
Searching the Web
CRISPR Cas9 mechanism clinical applications 2024 2025 breakthroughs
Reading File
Reading File
Reading File
Reading File
Running Command
mkdir -p /home/daytona/workspace/crispr-pptx && ls /home/daytona/workspace/crispr-pptx
Writing File
~/crispr-pptx/build.js
const pptxgen = require("pptxgenjs");
let pres = new pptxgen();
pres.layout = 'LAYOUT_16x9';
pres.title = 'CRISPR-Cas9: Genome Editing Technology';
pres.author = 'Biochemistry Seminar - 1st Year MBBS';
// ─── COLOR PALETTE ──────────────────────────────────────────────
// Deep navy dominates, electric teal accent, light slate text
const C = {
navy: "0D1B2A", // dominant background
darkNavy: "060D14", // darker variant for sections
teal: "00C2A0", // accent / headings
tealLight: "00E5BF", // lighter teal for highlights
white: "FFFFFF",
offWhite: "E8F4F2",
slate: "A8C5C0", // muted body text
coral: "FF6B6B", // warning / limitations
gold: "FFD166", // emphasis
charcoal: "1E3040", // card backgrounds
};
// ─── HELPER FUNCTIONS ───────────────────────────────────────────
function addSlideTitle(slide, title, subtitle) {
// Full-width teal bar at top
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fontFace: "Calibri", valign: "middle", margin: 0
});
if (subtitle) {
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fontSize: 12, color: C.tealLight,
fontFace: "Calibri", italic: true, valign: "middle", margin: 0
});
}
}
function bullet(text, sub) {
return { text, options: { bullet: { code: "2022" }, color: C.offWhite, fontSize: 15, fontFace: "Calibri", indentLevel: sub ? 1 : 0, breakLine: true } };
}
function subbullet(text) { return bullet(text, true); }
function addCard(slide, x, y, w, h, title, lines) {
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x, y, w, h,
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slide.addText(lines, {
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fontSize: 11, color: C.offWhite,
fontFace: "Calibri", valign: "top", margin: 0
});
}
// ════════════════════════════════════════════════════════════════
// SLIDE 1 — TITLE SLIDE
// ════════════════════════════════════════════════════════════════
let s1 = pres.addSlide();
s1.background = { color: C.navy };
// Large teal accent band
s1.addShape(pres.ShapeType.rect, {
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s1.addShape(pres.ShapeType.rect, {
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s1.addText("CRISPR-Cas9", {
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});
s1.addText("Genome Editing Technology", {
x: 0.5, y: 1.55, w: 9, h: 0.4,
fontSize: 22, color: C.offWhite,
fontFace: "Calibri", align: "center"
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s1.addText("Biochemistry Seminar | 1st Year MBBS", {
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fontSize: 14, color: C.slate,
fontFace: "Calibri", align: "center", italic: true
});
// DNA helix decorative dots
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});
}
s1.addText([
{ text: "Nobel Prize in Chemistry 2020 | ", options: { color: C.gold, fontSize: 12 } },
{ text: "Doudna & Charpentier", options: { color: C.tealLight, fontSize: 12, bold: true } }
], { x: 0.5, y: 4.3, w: 9, h: 0.3, align: "center" });
s1.addText([
{ text: "Sources: ", options: { bold: true, color: C.slate, fontSize: 10 } },
{ text: "Harper's Biochemistry 32e | Schwartz's Surgery 11e | Tietz Lab Medicine 7e", options: { color: C.slate, fontSize: 10 } }
], { x: 0.5, y: 5.2, w: 9, h: 0.3, align: "center" });
// ════════════════════════════════════════════════════════════════
// SLIDE 2 — OUTLINE
// ════════════════════════════════════════════════════════════════
let s2 = pres.addSlide();
s2.background = { color: C.navy };
addSlideTitle(s2, "Seminar Outline", "What we will cover today");
const topics = [
["01", "Historical background & discovery"],
["02", "Biological origin — bacterial immune system"],
["03", "Molecular components of CRISPR-Cas9"],
["04", "Mechanism of action (step-by-step)"],
["05", "DNA repair pathways: NHEJ vs HDR"],
["06", "Delivery methods"],
["07", "Medical & clinical applications"],
["08", "Limitations & off-target effects"],
["09", "Ethical considerations"],
["10", "Future perspectives & FDA approvals"],
];
const col1 = topics.slice(0, 5);
const col2 = topics.slice(5);
col1.forEach(([num, label], i) => {
s2.addShape(pres.ShapeType.roundRect, {
x: 0.3, y: 0.9 + i * 0.9, w: 0.45, h: 0.45,
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col2.forEach(([num, label], i) => {
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s2.addText(label, {
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fontSize: 13, color: C.offWhite,
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});
});
// ════════════════════════════════════════════════════════════════
// SLIDE 3 — HISTORY & DISCOVERY
// ════════════════════════════════════════════════════════════════
let s3 = pres.addSlide();
s3.background = { color: C.navy };
addSlideTitle(s3, "History & Discovery", "From bacterial genome curiosity to Nobel Prize");
const timeline = [
{ year: "1987", color: C.slate, text: "CRISPR repeats first described in E. coli genome by Yoshizumi Ishino (Japan)" },
{ year: "2002", color: C.tealLight, text: "Term 'CRISPR' coined; Cas genes identified flanking the repeat arrays" },
{ year: "2007", color: C.tealLight, text: "Barrangou et al. demonstrate CRISPR provides adaptive immunity in bacteria (Streptococcus thermophilus)" },
{ year: "2012", color: C.teal, text: "Doudna & Charpentier show Cas9 + guide RNA can cut purified DNA precisely (Science)" },
{ year: "2013", color: C.gold, text: "Zhang (Broad Institute) & Church (Harvard) adapt CRISPR-Cas9 for mammalian cell editing" },
{ year: "2020", color: C.coral, text: "Nobel Prize in Chemistry awarded to Jennifer Doudna & Emmanuelle Charpentier" },
{ year: "2023", color: C.teal, text: "FDA approves CASGEVY — first CRISPR-based therapy — for sickle cell disease & β-thalassemia" },
];
timeline.forEach((item, i) => {
let yPos = 0.95 + i * 0.66;
// Year pill
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x: 0.25, y: yPos, w: 0.75, h: 0.38,
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s3.addText(item.year, {
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fontSize: 11, bold: true, color: item.color,
fontFace: "Calibri", align: "center", valign: "middle", margin: 0
});
// Connector dot
s3.addShape(pres.ShapeType.ellipse, {
x: 1.1, y: yPos + 0.14, w: 0.12, h: 0.12,
fill: { color: item.color }, line: { type: "none" }
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// Text
s3.addText(item.text, {
x: 1.3, y: yPos, w: 8.4, h: 0.38,
fontSize: 12, color: C.offWhite,
fontFace: "Calibri", valign: "middle", margin: 0
});
// Vertical line connector (except last)
if (i < timeline.length - 1) {
s3.addShape(pres.ShapeType.line, {
x: 0.595, y: yPos + 0.38, w: 0, h: 0.28,
line: { color: C.slate, pt: 1, dashType: "dash" }
});
}
});
// ════════════════════════════════════════════════════════════════
// SLIDE 4 — BACTERIAL IMMUNE SYSTEM
// ════════════════════════════════════════════════════════════════
let s4 = pres.addSlide();
s4.background = { color: C.navy };
addSlideTitle(s4, "Biological Origin: Bacterial Adaptive Immunity", "How bacteria fight viruses using CRISPR");
s4.addText("CRISPR = Clustered Regularly Interspaced Short Palindromic Repeats", {
x: 0.3, y: 0.85, w: 9.4, h: 0.4,
fontSize: 14, bold: true, color: C.gold,
fontFace: "Calibri", align: "center"
});
const phases = [
{
icon: "①",
title: "ADAPTATION PHASE",
color: C.teal,
text: "Invading phage DNA fragment ('proto-spacer') is inserted into the bacterial CRISPR locus as a new spacer. This creates a 'memory' of the infection. PAM sequence (NGG) marks the proto-spacer."
},
{
icon: "②",
title: "crRNA BIOGENESIS",
color: C.gold,
text: "The CRISPR array is transcribed into a long pre-crRNA. Cas proteins cleave it into short CRISPR RNAs (crRNAs), each containing one spacer sequence. A transactivating crRNA (tracrRNA) base-pairs with crRNA."
},
{
icon: "③",
title: "INTERFERENCE PHASE",
color: C.coral,
text: "On re-exposure, crRNA–tracrRNA–Cas9 complex scans DNA. The crRNA base-pairs with complementary foreign DNA. Cas9 introduces a double-strand break (DSB), destroying the invader's genome."
},
];
phases.forEach((p, i) => {
let xPos = 0.25 + i * 3.22;
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x: xPos, y: 1.4, w: 3.1, h: 3.8,
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s4.addText(p.icon, {
x: xPos, y: 1.45, w: 3.1, h: 0.5,
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});
s4.addText(p.title, {
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if (i < 2) {
s4.addText("→", {
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}
});
s4.addText("Source: Tietz Textbook of Lab Medicine 7e, Chapter 67 | Schwartz's Surgery 11e, Chapter 15", {
x: 0.3, y: 5.3, w: 9.4, h: 0.25,
fontSize: 9, color: C.slate, fontFace: "Calibri", italic: true
});
// ════════════════════════════════════════════════════════════════
// SLIDE 5 — MOLECULAR COMPONENTS
// ════════════════════════════════════════════════════════════════
let s5 = pres.addSlide();
s5.background = { color: C.navy };
addSlideTitle(s5, "Molecular Components of CRISPR-Cas9", "Two-component system: Cas9 protein + guide RNA");
// Left column — Cas9 protein
s5.addShape(pres.ShapeType.roundRect, {
x: 0.25, y: 0.9, w: 4.55, h: 4.5,
fill: { color: C.charcoal }, line: { color: C.teal, pt: 2 }, rectRadius: 0.1
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s5.addText("CAS9 PROTEIN", {
x: 0.35, y: 0.95, w: 4.35, h: 0.38,
fontSize: 14, bold: true, color: C.teal,
fontFace: "Calibri", align: "center", margin: 0
});
s5.addText("(from Streptococcus pyogenes)", {
x: 0.35, y: 1.3, w: 4.35, h: 0.28,
fontSize: 10, color: C.slate, italic: true,
fontFace: "Calibri", align: "center", margin: 0
});
const cas9Points = [
["RuvC-like domain", "Cuts the non-template (proto-spacer) strand"],
["HNH-like domain", "Cuts the template strand (complementary to guide RNA)"],
["PAM interaction", "Reads NGG motif to position cleavage — 3 bp upstream of cut"],
["Blunt cut", "Generates a blunt-ended DSB 3 bp 5' to the PAM sequence"],
["Size", "~160 kDa; ~1368 aa; bilobed architecture"],
];
cas9Points.forEach(([title, desc], i) => {
let yy = 1.68 + i * 0.7;
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s5.addText([
{ text: title + ": ", options: { bold: true, color: C.tealLight, fontSize: 12 } },
{ text: desc, options: { color: C.offWhite, fontSize: 11 } }
], { x: 0.6, y: yy, w: 4.1, h: 0.6, fontFace: "Calibri", valign: "top", margin: 0 });
});
// Right column — Guide RNA
s5.addShape(pres.ShapeType.roundRect, {
x: 5.2, y: 0.9, w: 4.55, h: 4.5,
fill: { color: C.charcoal }, line: { color: C.gold, pt: 2 }, rectRadius: 0.1
});
s5.addText("GUIDE RNA (sgRNA)", {
x: 5.3, y: 0.95, w: 4.35, h: 0.38,
fontSize: 14, bold: true, color: C.gold,
fontFace: "Calibri", align: "center", margin: 0
});
s5.addText("(single guide RNA = crRNA + tracrRNA fusion)", {
x: 5.3, y: 1.3, w: 4.35, h: 0.28,
fontSize: 10, color: C.slate, italic: true,
fontFace: "Calibri", align: "center", margin: 0
});
const rnaPoints = [
["Spacer sequence", "20 nt complementary to target DNA; determines specificity"],
["tracrRNA scaffold", "Structural backbone; binds and activates Cas9"],
["Loop region", "Connects spacer to scaffold in sgRNA design"],
["Programmable", "Only the 20 nt spacer needs to be changed per target"],
["Off-target risk", "Mismatches tolerated up to ~5 nt from PAM-distal end"],
];
rnaPoints.forEach(([title, desc], i) => {
let yy = 1.68 + i * 0.7;
s5.addShape(pres.ShapeType.ellipse, { x: 5.33, y: yy + 0.08, w: 0.16, h: 0.16, fill: { color: C.gold }, line: { type: "none" } });
s5.addText([
{ text: title + ": ", options: { bold: true, color: C.gold, fontSize: 12 } },
{ text: desc, options: { color: C.offWhite, fontSize: 11 } }
], { x: 5.55, y: yy, w: 4.1, h: 0.6, fontFace: "Calibri", valign: "top", margin: 0 });
});
s5.addText("Source: Schwartz's Surgery 11e, pp. 534–536 | Harper's Biochemistry 32e, Table 39-2", {
x: 0.3, y: 5.35, w: 9.4, h: 0.25,
fontSize: 9, color: C.slate, fontFace: "Calibri", italic: true
});
// ════════════════════════════════════════════════════════════════
// SLIDE 6 — MECHANISM OF ACTION
// ════════════════════════════════════════════════════════════════
let s6 = pres.addSlide();
s6.background = { color: C.navy };
addSlideTitle(s6, "Mechanism of Action", "Step-by-step guide RNA-directed genome cleavage");
const steps = [
{ n: "1", title: "Guide RNA Design", color: C.teal, text: "A 20-nucleotide spacer sequence is designed complementary to the target DNA locus. This is cloned into an expression vector upstream of the tracrRNA scaffold to form sgRNA." },
{ n: "2", title: "Complex Assembly", color: C.tealLight, text: "Cas9 protein binds the sgRNA scaffold. The complex undergoes conformational change that activates the nuclease domains." },
{ n: "3", title: "PAM Recognition", color: C.gold, text: "The complex scans DNA for the PAM motif (5'-NGG-3'). On finding it, Cas9 unwinds the dsDNA and checks for complementarity between sgRNA and target strand." },
{ n: "4", title: "R-loop Formation", color: C.gold, text: "If sgRNA matches the target (seed region: 12 nt proximal to PAM must match), the sgRNA invades the DNA and forms a stable R-loop hybrid." },
{ n: "5", title: "Double-Strand Break", color: C.coral, text: "HNH cleaves the target strand; RuvC cleaves the non-target strand. Result: blunt-ended DSB 3 bp upstream of the PAM site." },
{ n: "6", title: "DNA Repair", color: C.coral, text: "Cell repairs DSB via NHEJ (error-prone → gene KO) or HDR (precise editing using a donor template). Outcome determines whether gene is disrupted or modified." },
];
steps.forEach((step, i) => {
let col = i < 3 ? 0 : 1;
let row = i % 3;
let xPos = 0.25 + col * 5.0;
let yPos = 0.88 + row * 1.56;
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fill: { color: C.charcoal }, line: { color: step.color, pt: 1.5 }, rectRadius: 0.08
});
s6.addShape(pres.ShapeType.ellipse, {
x: xPos + 0.1, y: yPos + 0.12, w: 0.45, h: 0.45,
fill: { color: step.color }, line: { type: "none" }
});
s6.addText(step.n, {
x: xPos + 0.1, y: yPos + 0.12, w: 0.45, h: 0.45,
fontSize: 16, bold: true, color: C.navy,
fontFace: "Calibri", align: "center", valign: "middle", margin: 0
});
s6.addText(step.title, {
x: xPos + 0.65, y: yPos + 0.1, w: 3.9, h: 0.35,
fontSize: 13, bold: true, color: step.color,
fontFace: "Calibri", valign: "middle", margin: 0
});
s6.addText(step.text, {
x: xPos + 0.12, y: yPos + 0.52, w: 4.46, h: 0.88,
fontSize: 10.5, color: C.offWhite,
fontFace: "Calibri", valign: "top", margin: 0
});
});
// ════════════════════════════════════════════════════════════════
// SLIDE 7 — NHEJ vs HDR
// ════════════════════════════════════════════════════════════════
let s7 = pres.addSlide();
s7.background = { color: C.navy };
addSlideTitle(s7, "DNA Repair Pathways After DSB", "NHEJ vs Homology-Directed Repair (HDR)");
// Shared intro
s7.addText("After Cas9-induced double-strand break (DSB), the cell repairs via one of two pathways:", {
x: 0.3, y: 0.83, w: 9.4, h: 0.32,
fontSize: 12, color: C.offWhite, fontFace: "Calibri"
});
// NHEJ card
s7.addShape(pres.ShapeType.roundRect, {
x: 0.25, y: 1.22, w: 4.55, h: 4.15,
fill: { color: C.charcoal }, line: { color: C.coral, pt: 2 }, rectRadius: 0.1
});
s7.addText("NON-HOMOLOGOUS END-JOINING (NHEJ)", {
x: 0.35, y: 1.27, w: 4.35, h: 0.5,
fontSize: 12, bold: true, color: C.coral,
fontFace: "Calibri", align: "center", margin: 0
});
const nhejPoints = [
"No repair template needed",
"Active in all cell cycle phases",
"End-binding proteins (Ku70/Ku80) recognize DSB",
"Ligation is error-prone → random indels",
"Frameshift mutations → premature stop codons",
"Result: GENE KNOCKOUT (KO)",
"High efficiency — preferred for loss-of-function studies",
];
nhejPoints.forEach((pt, i) => {
s7.addShape(pres.ShapeType.ellipse, { x: 0.38, y: 1.85 + i * 0.48, w: 0.13, h: 0.13, fill: { color: C.coral }, line: { type: "none" } });
s7.addText(pt, {
x: 0.58, y: 1.82 + i * 0.48, w: 4.1, h: 0.38,
fontSize: 11, color: i === 5 ? C.coral : C.offWhite,
bold: i === 5, fontFace: "Calibri", valign: "middle", margin: 0
});
});
// HDR card
s7.addShape(pres.ShapeType.roundRect, {
x: 5.2, y: 1.22, w: 4.55, h: 4.15,
fill: { color: C.charcoal }, line: { color: C.teal, pt: 2 }, rectRadius: 0.1
});
s7.addText("HOMOLOGY-DIRECTED REPAIR (HDR)", {
x: 5.3, y: 1.27, w: 4.35, h: 0.5,
fontSize: 12, bold: true, color: C.teal,
fontFace: "Calibri", align: "center", margin: 0
});
const hdrPoints = [
"Requires homologous donor template (ssDNA or dsDNA)",
"Active mainly in S/G2 phase → lower efficiency",
"Uses sequence from template to repair DSB",
"Precise insertion, deletion, or point mutation",
"Can add reporter genes (GFP, luciferase) or tags",
"Result: PRECISE GENE EDITING",
"Lower efficiency than NHEJ; cell-type dependent",
];
hdrPoints.forEach((pt, i) => {
s7.addShape(pres.ShapeType.ellipse, { x: 5.33, y: 1.85 + i * 0.48, w: 0.13, h: 0.13, fill: { color: C.teal }, line: { type: "none" } });
s7.addText(pt, {
x: 5.53, y: 1.82 + i * 0.48, w: 4.1, h: 0.38,
fontSize: 11, color: i === 5 ? C.tealLight : C.offWhite,
bold: i === 5, fontFace: "Calibri", valign: "middle", margin: 0
});
});
s7.addText("Source: Schwartz's Surgery 11e, pp. 533–534", {
x: 0.3, y: 5.35, w: 9.4, h: 0.25,
fontSize: 9, color: C.slate, fontFace: "Calibri", italic: true
});
// ════════════════════════════════════════════════════════════════
// SLIDE 8 — MEDICAL APPLICATIONS
// ════════════════════════════════════════════════════════════════
let s8 = pres.addSlide();
s8.background = { color: C.navy };
addSlideTitle(s8, "Medical Applications & Clinical Trials", "From bench to bedside");
const apps = [
{
cat: "HAEMATOLOGY",
color: C.coral,
items: [
"CASGEVY (exagamglogene autotemcel) — FDA approved Dec 2023",
"Sickle cell disease: reactivates fetal haemoglobin (HbF) by disrupting BCL11A gene",
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"95.6% of SCD patients free from vaso-occlusive crises for ≥12 months"
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"Universal donor CAR-T cells: off-the-shelf cancer immunotherapy"
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"COVID-19: SHERLOCK (CRISPR-Cas13) diagnostic assay received FDA EUA"
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"Hereditary angioedema: CRISPR KO of KLKB1 gene in liver — single IV dose",
"CPS1 deficiency: personalised CRISPR therapy in infant (lipid nanoparticle delivery, May 2025)",
"Duchenne muscular dystrophy: exon skipping to restore dystrophin reading frame"
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// ════════════════════════════════════════════════════════════════
// SLIDE 9 — LIMITATIONS & OFF-TARGET EFFECTS
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// ════════════════════════════════════════════════════════════════
// SLIDE 10 — ETHICAL CONSIDERATIONS
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"LMICs excluded from clinical trial recruitment and benefit"
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// ════════════════════════════════════════════════════════════════
// SLIDE 11 — FUTURE PERSPECTIVES & FDA APPROVALS
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{ phase: "Phase II", drug: "EDIT-301", cond: "SCD / β-Thalassaemia", detail: "HbF reactivation via AsCas12a editing of HBG1/2 promoters" },
{ phase: "Phase I/II", drug: "EBT-101", cond: "HIV/AIDS", detail: "Excision Biotherapeutics — excision of HIV proviral DNA" },
{ phase: "Phase I/II", drug: "NTLA-5001", cond: "Acute Myeloid Leukemia", detail: "CRISPR-edited T-cell receptor T-cells targeting WT1 antigen" },
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{ tool: "CRISPRi / CRISPRa", desc: "dCas9 fused to repressors/activators; controls gene expression without cuts" },
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// ════════════════════════════════════════════════════════════════
// SLIDE 12 — SUMMARY / KEY TAKEAWAYS
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// ────────────────────────────────────────────────────────────────
// WRITE FILE
// ────────────────────────────────────────────────────────────────
pres.writeFile({ fileName: "/home/daytona/workspace/crispr-pptx/CRISPR_Cas9_MBBS_Seminar.pptx" })
.then(() => console.log("✓ Presentation written successfully"))
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Running Command
cd /home/daytona/workspace/crispr-pptx && node build.js 2>&1
Running Command
ls -lh /home/daytona/workspace/crispr-pptx/CRISPR_Cas9_MBBS_Seminar.pptx
Your presentation is ready! Download it here:
CRISPR Cas9 MBBS Seminar
Presentation · PPTX
What's in the deck (12 slides)
| # | Slide | Highlights |
|---|---|---|
| 1 | Title Slide | Dark navy + electric teal design; Nobel Prize callout |
| 2 | Outline | All 10 topics listed with numbered cards |
| 3 | History & Discovery | Timeline from 1987 E. coli discovery → 2023 FDA approval |
| 4 | Bacterial Immune System | 3-phase process (Adaptation → crRNA biogenesis → Interference) |
| 5 | Molecular Components | Cas9 domains (RuvC, HNH, PAM) vs sgRNA structure, side-by-side |
| 6 | Mechanism of Action | 6-step process: guide design → PAM recognition → R-loop → DSB |
| 7 | NHEJ vs HDR | Dual card layout comparing both repair pathways with clinical relevance |
| 8 | Medical Applications | 4 categories: Haematology, Oncology, Infectious Disease, Metabolic/Neuro |
| 9 | Limitations | 6 challenges: off-targets, delivery, mosaicism, HDR inefficiency, immunogenicity, cost |
| 10 | Ethics | Germline editing (He Jiankui scandal), Access & Equity, Consent issues |
| 11 | Future & FDA Approvals | CASGEVY approval details, pipeline drugs, next-gen tools (Base/Prime editing) |
| 12 | Key Takeaways | 5 numbered summary points |
Sources used:
- Harper's Illustrated Biochemistry 32e (Table 39-2)
- Schwartz's Principles of Surgery 11e (Chapter 15, pp. 533-536)
- Tietz Textbook of Laboratory Medicine 7e (Chapter 67)
- PubMed: Tyumentseva 2023, Laurent 2024, Singh 2024, Cetin 2025
- CRISPR Medicine News (June 2025 - CASGEVY 5.5-year follow-up data)
Can you do it again
Asking for Preferences
Asking for Preferences
Reading File
Writing File
~/crispr-pptx/build_v2.js
const pptxgen = require("pptxgenjs");
let pres = new pptxgen();
pres.layout = 'LAYOUT_16x9';
pres.title = 'CRISPR-Cas9: Genome Editing Technology';
// ─── PALETTE: dark green + lime biotech ────────────────────────
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// ════════════════════════════════════════════════════════════════
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x: 1.5, y: 2.1, w: 7.0, h: 0.06,
fill: { color: C.green }, line: { type: "none" }
});
s.addText("Cas9 Gene Editing Technology", {
x: 0.4, y: 2.22, w: 9.2, h: 0.55,
fontSize: 26, bold: false, color: C.offWhite,
fontFace: "Trebuchet MS", align: "center", charSpacing: 2, margin: 0
});
s.addText("Biochemistry Seminar · 1st Year MBBS", {
x: 0.4, y: 2.88, w: 9.2, h: 0.38,
fontSize: 15, color: C.muted, italic: true,
fontFace: "Trebuchet MS", align: "center", margin: 0
});
// Nobel callout pill
s.addShape(pres.ShapeType.roundRect, {
x: 2.8, y: 3.5, w: 4.4, h: 0.55,
fill: { color: C.bgPanel }, line: { color: C.amber, pt: 1.5 }, rectRadius: 0.28
});
s.addText("🏆 Nobel Prize in Chemistry 2020 — Doudna & Charpentier", {
x: 2.8, y: 3.5, w: 4.4, h: 0.55,
fontSize: 11, bold: true, color: C.amber,
fontFace: "Trebuchet MS", align: "center", valign: "middle", margin: 0
});
// DNA dots decoration
const dotsY = [4.3, 4.5, 4.3, 4.5, 4.3, 4.5, 4.3, 4.5, 4.3, 4.5, 4.3, 4.5];
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s.addShape(pres.ShapeType.ellipse, {
x: 0.5 + i * 0.76, y: dotsY[i], w: 0.16, h: 0.16,
fill: { color: i % 2 === 0 ? C.lime : C.green }, line: { type: "none" }
});
if (i < 11) {
s.addShape(pres.ShapeType.line, {
x: 0.66 + i * 0.76, y: dotsY[i] + 0.08, w: 0.6, h: (dotsY[i+1] - dotsY[i]),
line: { color: C.muted, pt: 1 }
});
}
}
}
// ════════════════════════════════════════════════════════════════
// SLIDE 2 — WHAT IS CRISPR? (intro + definition)
// ════════════════════════════════════════════════════════════════
{
let s = pres.addSlide();
s.background = { color: C.bg };
header(s, "What is CRISPR-Cas9?", "A programmable molecular scissors for genome editing");
// Large acronym breakdown
const letters = [
{ L: "C", word: "Clustered" },
{ L: "R", word: "Regularly" },
{ L: "I", word: "Interspaced" },
{ L: "S", word: "Short" },
{ L: "P", word: "Palindromic" },
{ L: "R", word: "Repeats" },
];
letters.forEach((item, i) => {
let xPos = 0.38 + i * 1.58;
s.addShape(pres.ShapeType.rect, {
x: xPos, y: 1.0, w: 1.45, h: 0.7,
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});
s.addText(item.L, {
x: xPos, y: 1.0, w: 1.45, h: 0.7,
fontSize: 32, bold: true, color: C.darkText,
fontFace: "Trebuchet MS", align: "center", valign: "middle", margin: 0
});
s.addText(item.word, {
x: xPos, y: 1.72, w: 1.45, h: 0.35,
fontSize: 10, color: C.offWhite, italic: true,
fontFace: "Trebuchet MS", align: "center", margin: 0
});
});
// Two info cards
card(s, 0.38, 2.2, 4.5, 3.1, C.lime);
s.addText("ORIGIN", {
x: 0.5, y: 2.28, w: 4.26, h: 0.32,
fontSize: 13, bold: true, color: C.lime,
fontFace: "Trebuchet MS", margin: 0
});
const originLines = [
"First described in E. coli genome in 1987 by Yoshizumi Ishino",
"Spacer sequences were later found to be fragments of past viral invaders — a form of bacterial 'immunological memory'",
"Cas (CRISPR-associated) proteins are nucleases encoded by genes flanking the CRISPR array",
"Adapted for eukaryotic genome editing in 2012–2013",
"Now the most widely used gene-editing tool in biology",
];
originLines.forEach((line, i) => {
s.addShape(pres.ShapeType.ellipse, { x: 0.5, y: 2.68 + i * 0.52, w: 0.12, h: 0.12, fill: { color: C.lime }, line: { type: "none" } });
s.addText(line, {
x: 0.7, y: 2.64 + i * 0.52, w: 4.08, h: 0.46,
fontSize: 11, color: C.offWhite,
fontFace: "Trebuchet MS", valign: "middle", margin: 0
});
});
card(s, 5.12, 2.2, 4.5, 3.1, C.green);
s.addText("WHY IT MATTERS", {
x: 5.24, y: 2.28, w: 4.26, h: 0.32,
fontSize: 13, bold: true, color: C.green,
fontFace: "Trebuchet MS", margin: 0
});
const whyLines = [
"Previous methods (RNAi, TALENs, ZFNs) were slow, costly, or imprecise",
"CRISPR-Cas9 needs only a 20 nt RNA change to redirect it to any gene",
"Can knock out, correct, insert or regulate any gene in any organism",
"Cheaper, faster, and more specific than any prior technology",
"First CRISPR therapy FDA-approved December 2023 (CASGEVY)",
];
whyLines.forEach((line, i) => {
s.addShape(pres.ShapeType.ellipse, { x: 5.24, y: 2.68 + i * 0.52, w: 0.12, h: 0.12, fill: { color: C.green }, line: { type: "none" } });
s.addText(line, {
x: 5.44, y: 2.64 + i * 0.52, w: 4.08, h: 0.46,
fontSize: 11, color: C.offWhite,
fontFace: "Trebuchet MS", valign: "middle", margin: 0
});
});
}
// ════════════════════════════════════════════════════════════════
// SLIDE 3 — HISTORY TIMELINE
// ════════════════════════════════════════════════════════════════
{
let s = pres.addSlide();
s.background = { color: C.bg };
header(s, "Discovery Timeline", "From bacterial curiosity to Nobel Prize to FDA approval");
const events = [
{ year: "1987", title: "First CRISPR Discovery", desc: "Yoshizumi Ishino (Japan) notices unusual repeat sequences in E. coli genome — significance unknown at the time", color: C.muted },
{ year: "2002", title: "CRISPR Named", desc: "Ruud Jansen coins 'CRISPR'; Cas genes identified flanking repeat arrays", color: C.green },
{ year: "2007", title: "Immune Function Proven", desc: "Barrangou et al. show CRISPR provides sequence-specific adaptive immunity in Streptococcus thermophilus against bacteriophages", color: C.green },
{ year: "2012", title: "Cas9 Programmable Cutter", desc: "Doudna & Charpentier (Science) demonstrate Cas9 + sgRNA can cut any purified DNA sequence at will", color: C.lime },
{ year: "2013", title: "Mammalian Cell Editing", desc: "Zhang (Broad) and Church (Harvard) independently achieve CRISPR editing in human cell lines", color: C.lime },
{ year: "2018", title: "He Jiankui Scandal", desc: "First CRISPR-edited human babies — internationally condemned as premature and unethical", color: C.red },
{ year: "2020", title: "Nobel Prize", desc: "Chemistry Nobel awarded to Jennifer Doudna & Emmanuelle Charpentier for CRISPR-Cas9", color: C.amber },
{ year: "2023", title: "FDA Approves CASGEVY", desc: "First CRISPR-based drug approved for sickle cell disease and β-thalassaemia", color: C.lime },
];
// Horizontal line
s.addShape(pres.ShapeType.rect, {
x: 0.38, y: 2.75, w: 9.3, h: 0.05,
fill: { color: C.border }, line: { type: "none" }
});
events.forEach((ev, i) => {
let xPos = 0.38 + i * 1.165;
let isAbove = i % 2 === 0;
// Dot on line
s.addShape(pres.ShapeType.ellipse, {
x: xPos + 0.37, y: 2.66, w: 0.22, h: 0.22,
fill: { color: ev.color }, line: { type: "none" }
});
if (isAbove) {
// Year above line
s.addText(ev.year, {
x: xPos, y: 1.68, w: 1.1, h: 0.3,
fontSize: 11, bold: true, color: ev.color,
fontFace: "Trebuchet MS", align: "center", margin: 0
});
// Vertical connector
s.addShape(pres.ShapeType.line, {
x: xPos + 0.485, y: 1.98, w: 0, h: 0.68,
line: { color: ev.color, pt: 1, dashType: "sysDash" }
});
// Card above
card(s, xPos, 0.92, 1.1, 0.76, ev.color);
s.addText(ev.title, {
x: xPos + 0.07, y: 0.95, w: 0.96, h: 0.32,
fontSize: 8.5, bold: true, color: ev.color,
fontFace: "Trebuchet MS", valign: "top", margin: 0
});
s.addText(ev.desc, {
x: xPos + 0.07, y: 1.24, w: 0.96, h: 0.4,
fontSize: 7.5, color: C.offWhite,
fontFace: "Trebuchet MS", valign: "top", margin: 0
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} else {
// Year below line
s.addText(ev.year, {
x: xPos, y: 2.94, w: 1.1, h: 0.3,
fontSize: 11, bold: true, color: ev.color,
fontFace: "Trebuchet MS", align: "center", margin: 0
});
s.addShape(pres.ShapeType.line, {
x: xPos + 0.485, y: 3.24, w: 0, h: 0.55,
line: { color: ev.color, pt: 1, dashType: "sysDash" }
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card(s, xPos, 3.79, 1.1, 1.55, ev.color);
s.addText(ev.title, {
x: xPos + 0.07, y: 3.83, w: 0.96, h: 0.32,
fontSize: 8.5, bold: true, color: ev.color,
fontFace: "Trebuchet MS", valign: "top", margin: 0
});
s.addText(ev.desc, {
x: xPos + 0.07, y: 4.12, w: 0.96, h: 1.16,
fontSize: 7.5, color: C.offWhite,
fontFace: "Trebuchet MS", valign: "top", margin: 0
});
}
});
}
// ════════════════════════════════════════════════════════════════
// SLIDE 4 — BACTERIAL IMMUNE SYSTEM (infographic)
// ════════════════════════════════════════════════════════════════
{
let s = pres.addSlide();
s.background = { color: C.bg };
header(s, "Biological Origin: Bacterial Adaptive Immunity", "CRISPR is nature's own antiviral defence system");
// Three-phase process with connecting arrows
const phases = [
{
num: "1",
title: "ADAPTATION",
color: C.green,
icon: "🦠",
steps: [
"Bacteriophage injects DNA into bacterium",
"Cas1 + Cas2 complex captures a short fragment (proto-spacer) of phage DNA",
"Proto-spacer must carry a PAM (NGG) motif",
"Fragment integrated into CRISPR array as a new SPACER",
"Cell now has immunological memory of this phage"
]
},
{
num: "2",
title: "crRNA BIOGENESIS",
color: C.lime,
icon: "🧪",
steps: [
"CRISPR array transcribed into long pre-crRNA",
"tracrRNA (trans-activating crRNA) base-pairs with pre-crRNA",
"RNase III cleaves into short crRNA fragments",
"Each crRNA contains ONE spacer sequence",
"crRNA + tracrRNA fused → single guide RNA (sgRNA) for lab use"
]
},
{
num: "3",
title: "INTERFERENCE",
color: C.amber,
icon: "✂",
steps: [
"Re-exposure to same phage triggers interference",
"crRNA–tracrRNA–Cas9 complex assembles",
"Scans incoming DNA for PAM motif",
"sgRNA base-pairs with complementary target strand",
"Cas9 HNH + RuvC domains cleave both strands → DSB"
]
}
];
phases.forEach((ph, i) => {
let xPos = 0.38 + i * 3.22;
// Card
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x: xPos, y: 0.95, w: 3.05, h: 4.45,
fill: { color: C.bgAlt }, line: { color: ph.color, pt: 2 }, rectRadius: 0.12
});
// Phase number circle
s.addShape(pres.ShapeType.ellipse, {
x: xPos + 0.12, y: 1.0, w: 0.52, h: 0.52,
fill: { color: ph.color }, line: { type: "none" }
});
s.addText(ph.num, {
x: xPos + 0.12, y: 1.0, w: 0.52, h: 0.52,
fontSize: 20, bold: true, color: C.darkText,
fontFace: "Trebuchet MS", align: "center", valign: "middle", margin: 0
});
s.addText(ph.title, {
x: xPos + 0.72, y: 1.03, w: 2.25, h: 0.45,
fontSize: 14, bold: true, color: ph.color,
fontFace: "Trebuchet MS", valign: "middle", margin: 0, charSpacing: 1
});
ph.steps.forEach((step, j) => {
s.addShape(pres.ShapeType.rect, {
x: xPos + 0.15, y: 1.62 + j * 0.58, w: 0.06, h: 0.36,
fill: { color: ph.color }, line: { type: "none" }
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s.addText(step, {
x: xPos + 0.28, y: 1.60 + j * 0.58, w: 2.66, h: 0.42,
fontSize: 10.5, color: C.offWhite,
fontFace: "Trebuchet MS", valign: "middle", margin: 0
});
});
// Arrow between phases
if (i < 2) {
s.addText("▶", {
x: xPos + 3.05, y: 2.9, w: 0.22, h: 0.4,
fontSize: 18, color: C.muted,
fontFace: "Trebuchet MS", align: "center", valign: "middle", margin: 0
});
}
});
s.addText("Source: Tietz Lab Medicine 7e, Ch. 67 | Schwartz's Surgery 11e, Ch. 15", {
x: 0.38, y: 5.4, w: 9.3, h: 0.2,
fontSize: 8.5, color: C.muted, italic: true, fontFace: "Trebuchet MS"
});
}
// ════════════════════════════════════════════════════════════════
// SLIDE 5 — MOLECULAR COMPONENTS (visual)
// ════════════════════════════════════════════════════════════════
{
let s = pres.addSlide();
s.background = { color: C.bg };
header(s, "Molecular Components", "Two-component system: Cas9 protein + single guide RNA");
// Central "scissor" diagram area
s.addShape(pres.ShapeType.roundRect, {
x: 3.8, y: 1.0, w: 2.4, h: 4.3,
fill: { color: C.bgPanel }, line: { color: C.lime, pt: 2 }, rectRadius: 0.12
});
s.addText("CAS9\n+\nsgRNA\nCOMPLEX", {
x: 3.8, y: 1.8, w: 2.4, h: 2.0,
fontSize: 18, bold: true, color: C.lime,
fontFace: "Trebuchet MS", align: "center", valign: "middle", margin: 0
});
s.addText("✂", {
x: 3.8, y: 3.85, w: 2.4, h: 0.8,
fontSize: 38, color: C.lime,
fontFace: "Trebuchet MS", align: "center", valign: "middle", margin: 0
});
// Connecting lines to cards
// Left to center
s.addShape(pres.ShapeType.line, { x: 3.2, y: 3.15, w: 0.6, h: 0, line: { color: C.lime, pt: 1.5, dashType: "dash" } });
// Right to center
s.addShape(pres.ShapeType.line, { x: 6.2, y: 3.15, w: 0.6, h: 0, line: { color: C.green, pt: 1.5, dashType: "dash" } });
// LEFT — Cas9 card
s.addShape(pres.ShapeType.roundRect, {
x: 0.28, y: 1.0, w: 3.2, h: 4.3,
fill: { color: C.bgAlt }, line: { color: C.lime, pt: 1.5 }, rectRadius: 0.1
});
s.addText("CAS9 PROTEIN", {
x: 0.38, y: 1.06, w: 3.0, h: 0.38,
fontSize: 13, bold: true, color: C.lime,
fontFace: "Trebuchet MS", align: "center", charSpacing: 1, margin: 0
});
s.addText("Streptococcus pyogenes (SpCas9)", {
x: 0.38, y: 1.42, w: 3.0, h: 0.26,
fontSize: 9.5, color: C.muted, italic: true,
fontFace: "Trebuchet MS", align: "center", margin: 0
});
const cas9 = [
["Size", "~160 kDa, 1368 amino acids"],
["Architecture", "Bilobed — recognition lobe + nuclease lobe"],
["RuvC domain", "Cleaves non-template (PAM-containing) strand"],
["HNH domain", "Cleaves template strand (sgRNA-complementary)"],
["PAM site", "Reads 5'-NGG-3'; positions Cas9 for cleavage"],
["Cut site", "Blunt-end DSB 3 bp upstream of PAM"],
["dCas9", "Dead Cas9 — no nuclease; used for CRISPRi/a"],
];
cas9.forEach(([k, v], i) => {
s.addText([
{ text: k + ": ", options: { bold: true, color: C.lime, fontSize: 10.5 } },
{ text: v, options: { color: C.offWhite, fontSize: 10.5 } }
], {
x: 0.38, y: 1.75 + i * 0.5, w: 3.0, h: 0.44,
fontFace: "Trebuchet MS", valign: "middle", margin: 0
});
});
// RIGHT — sgRNA card
s.addShape(pres.ShapeType.roundRect, {
x: 6.52, y: 1.0, w: 3.2, h: 4.3,
fill: { color: C.bgAlt }, line: { color: C.green, pt: 1.5 }, rectRadius: 0.1
});
s.addText("SINGLE GUIDE RNA (sgRNA)", {
x: 6.62, y: 1.06, w: 3.0, h: 0.38,
fontSize: 13, bold: true, color: C.green,
fontFace: "Trebuchet MS", align: "center", charSpacing: 1, margin: 0
});
s.addText("crRNA + tracrRNA fusion", {
x: 6.62, y: 1.42, w: 3.0, h: 0.26,
fontSize: 9.5, color: C.muted, italic: true,
fontFace: "Trebuchet MS", align: "center", margin: 0
});
const sgrna = [
["Spacer", "20 nt complementary to target DNA — determines specificity"],
["Scaffold", "tracrRNA-derived; binds and activates Cas9"],
["Seed region", "12 nt PAM-proximal — must match for cleavage"],
["PAM-distal", "Up to 5 mismatches tolerated → off-target risk"],
["Design", "Only 20 nt change needed to retarget Cas9"],
["Delivery", "Plasmid, in vitro transcript, or synthetic RNA"],
["Off-target", "CCLMoff AI predicts off-target risk (2025 tool)"],
];
sgrna.forEach(([k, v], i) => {
s.addText([
{ text: k + ": ", options: { bold: true, color: C.green, fontSize: 10.5 } },
{ text: v, options: { color: C.offWhite, fontSize: 10.5 } }
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x: 6.62, y: 1.75 + i * 0.5, w: 3.0, h: 0.44,
fontFace: "Trebuchet MS", valign: "middle", margin: 0
});
});
s.addText("Source: Harper's Biochemistry 32e, Table 39-2 | Schwartz's Surgery 11e, p. 534", {
x: 0.38, y: 5.4, w: 9.3, h: 0.2,
fontSize: 8.5, color: C.muted, italic: true, fontFace: "Trebuchet MS"
});
}
// ════════════════════════════════════════════════════════════════
// SLIDE 6 — MECHANISM (step flow)
// ════════════════════════════════════════════════════════════════
{
let s = pres.addSlide();
s.background = { color: C.bg };
header(s, "Mechanism of Action", "How CRISPR-Cas9 finds and cuts its target");
const steps = [
{ n: "1", title: "Design sgRNA", color: C.green, text: "Select a 20 nt sequence from target locus immediately upstream of an NGG PAM motif. Use online tools (Benchling, CRISPOR) to minimise off-target risk. Clone into expression vector." },
{ n: "2", title: "Cas9 Loading", color: C.limeDeep, text: "Cas9 is expressed or introduced as protein/mRNA. It binds the sgRNA scaffold and undergoes conformational activation. The bilobed structure opens to accept the guide." },
{ n: "3", title: "DNA Scanning", color: C.lime, text: "Cas9–sgRNA complex diffuses along DNA. It pauses at each NGG PAM and locally unwinds dsDNA to check for sgRNA–target complementarity (3D sliding/hopping)." },
{ n: "4", title: "R-loop Formation", color: C.lime, text: "If the 20 nt seed region matches, the sgRNA invades the DNA duplex, displacing one strand to form a stable RNA–DNA hybrid (R-loop). This triggers nuclease domain activation." },
{ n: "5", title: "Double-Strand Break", color: C.amber, text: "HNH domain cleaves the template strand; RuvC domain cleaves the non-template strand. Result: blunt-ended DSB precisely 3 bp upstream of the PAM sequence." },
{ n: "6", title: "Cell Repair Response", color: C.red, text: "NHEJ: fast but error-prone; joins cut ends with random insertions/deletions → frameshift → gene KO. HDR: precise repair using a donor template → correction or insertion of any sequence." },
];
const cols = [steps.slice(0, 3), steps.slice(3)];
cols.forEach((col, ci) => {
col.forEach((step, ri) => {
let xPos = 0.28 + ci * 5.0;
let yPos = 0.95 + ri * 1.56;
s.addShape(pres.ShapeType.roundRect, {
x: xPos, y: yPos, w: 4.7, h: 1.46,
fill: { color: C.bgAlt }, line: { color: step.color, pt: 1.5 }, rectRadius: 0.1
});
// Step badge
s.addShape(pres.ShapeType.ellipse, {
x: xPos + 0.12, y: yPos + 0.1, w: 0.44, h: 0.44,
fill: { color: step.color }, line: { type: "none" }
});
s.addText(step.n, {
x: xPos + 0.12, y: yPos + 0.1, w: 0.44, h: 0.44,
fontSize: 18, bold: true, color: C.darkText,
fontFace: "Trebuchet MS", align: "center", valign: "middle", margin: 0
});
s.addText(step.title, {
x: xPos + 0.65, y: yPos + 0.1, w: 3.9, h: 0.38,
fontSize: 13, bold: true, color: step.color,
fontFace: "Trebuchet MS", valign: "middle", margin: 0
});
s.addText(step.text, {
x: xPos + 0.12, y: yPos + 0.56, w: 4.46, h: 0.84,
fontSize: 10.5, color: C.offWhite,
fontFace: "Trebuchet MS", valign: "top", margin: 0
});
// Down arrow between cards in same column
if (ri < 2) {
s.addText("▼", {
x: xPos + 1.9, y: yPos + 1.48, w: 0.5, h: 0.1,
fontSize: 10, color: C.muted,
fontFace: "Trebuchet MS", align: "center", margin: 0
});
}
});
});
}
// ════════════════════════════════════════════════════════════════
// SLIDE 7 — NHEJ vs HDR (split visual)
// ════════════════════════════════════════════════════════════════
{
let s = pres.addSlide();
s.background = { color: C.bg };
header(s, "DNA Repair After the Cut: NHEJ vs HDR", "The pathway chosen determines the biological outcome");
s.addText("Cas9 DSB", {
x: 4.05, y: 0.92, w: 1.9, h: 0.35,
fontSize: 12, bold: true, color: C.amber,
fontFace: "Trebuchet MS", align: "center", margin: 0
});
s.addShape(pres.ShapeType.ellipse, {
x: 4.6, y: 1.28, w: 0.8, h: 0.28,
fill: { color: C.amber }, line: { type: "none" }
});
// Left arrow
s.addText("◀─────", {
x: 0.28, y: 1.3, w: 4.2, h: 0.28,
fontSize: 13, color: C.red,
fontFace: "Trebuchet MS", align: "right", margin: 0
});
// Right arrow
s.addText("─────▶", {
x: 5.5, y: 1.3, w: 4.2, h: 0.28,
fontSize: 13, color: C.lime,
fontFace: "Trebuchet MS", align: "left", margin: 0
});
s.addText("No template available", {
x: 0.28, y: 1.62, w: 4.2, h: 0.25,
fontSize: 10, color: C.red, italic: true,
fontFace: "Trebuchet MS", align: "center", margin: 0
});
s.addText("Donor template provided", {
x: 5.5, y: 1.62, w: 4.2, h: 0.25,
fontSize: 10, color: C.lime, italic: true,
fontFace: "Trebuchet MS", align: "center", margin: 0
});
// NHEJ card
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x: 0.28, y: 1.92, w: 4.35, h: 3.4,
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});
s.addText("NHEJ", {
x: 0.38, y: 1.97, w: 4.15, h: 0.42,
fontSize: 18, bold: true, color: C.red,
fontFace: "Trebuchet MS", align: "center", charSpacing: 3, margin: 0
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s.addText("Non-Homologous End-Joining", {
x: 0.38, y: 2.36, w: 4.15, h: 0.3,
fontSize: 10, color: C.muted, italic: true,
fontFace: "Trebuchet MS", align: "center", margin: 0
});
const nhej = [
["Speed", "Fast; active throughout all cell cycle phases"],
["Template", "None required"],
["Mechanism", "Ku70/Ku80 bind DSB ends; DNAPK recruits ligase IV → Artmeis → ligates with errors"],
["Outcome", "Random indels (insertions/deletions) at cut site"],
["Effect", "Frameshift → premature stop codon → loss-of-function (gene KO)"],
["Use", "High-efficiency gene knockout experiments"],
["Efficiency", "Very high (~80–95% editing rate in many cell lines)"],
];
nhej.forEach(([k, v], i) => {
s.addText([
{ text: k + ": ", options: { bold: true, color: C.red, fontSize: 10.5 } },
{ text: v, options: { color: C.offWhite, fontSize: 10.5 } }
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fontFace: "Trebuchet MS", valign: "middle", margin: 0
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});
// HDR card
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x: 5.37, y: 1.92, w: 4.35, h: 3.4,
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s.addText("HDR", {
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s.addText("Homology-Directed Repair", {
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const hdr = [
["Speed", "Slow; restricted to S/G2 phase of cell cycle"],
["Template", "ssDNA oligo or dsDNA donor required"],
["Mechanism", "Broken strands invade donor template; DNA synthesis using template sequence"],
["Outcome", "Precise gene correction, insertion, or mutation"],
["Effect", "Any defined sequence change at exact location"],
["Use", "Therapeutic correction of monogenic diseases"],
["Efficiency", "Low (~1–10% in somatic cells; higher in iPSCs)"],
];
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{ text: k + ": ", options: { bold: true, color: C.lime, fontSize: 10.5 } },
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s.addText("Source: Schwartz's Principles of Surgery 11e, pp. 533–534", {
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fontSize: 8.5, color: C.muted, italic: true, fontFace: "Trebuchet MS"
});
}
// ════════════════════════════════════════════════════════════════
// SLIDE 8 — DELIVERY METHODS
// ════════════════════════════════════════════════════════════════
{
let s = pres.addSlide();
s.background = { color: C.bg };
header(s, "Delivery Methods", "How CRISPR components are introduced into target cells");
const methods = [
{
name: "Plasmid DNA",
color: C.green,
pros: ["Easy to prepare", "Encodes both Cas9 + sgRNA", "Long-term expression possible"],
cons: ["Risk of genomic integration", "Persistent expression → more off-targets", "Low efficiency in primary cells"]
},
{
name: "Viral Vectors",
color: C.lime,
pros: ["AAV: high in vivo tropism", "Lentivirus: stable integration", "High transduction efficiency"],
cons: ["AAV: 4.7 kb cargo limit (SpCas9 = 4.2 kb, tight fit)", "Immunogenicity risk", "Insertional mutagenesis (lentivirus)"]
},
{
name: "mRNA + sgRNA",
color: C.amber,
pros: ["No genomic integration risk", "Transient expression → fewer off-targets", "Suitable for LNP delivery"],
cons: ["mRNA instability; needs cold chain", "Less efficient in non-dividing cells", "Repeated dosing may be needed"]
},
{
name: "RNP Complex",
color: C.limeDeep,
pros: ["Cas9 protein + sgRNA pre-formed", "Immediate activity; rapidly degraded", "Lowest off-target rate of all methods"],
cons: ["Short activity window may reduce editing efficiency", "Expensive protein production at scale", "Limited in vivo delivery options"]
},
{
name: "Lipid Nanoparticles (LNP)",
color: C.green,
pros: ["Clinically validated (mRNA COVID vaccines)", "CPS1 infant case (2025): LNP delivered in vivo", "No immunogenicity from viral proteins"],
cons: ["Primarily liver-tropic; hard to retarget other tissues", "Endosomal escape efficiency limits potency", "Stability and storage challenges"]
},
{
name: "Electroporation / Nucleofection",
color: C.lime,
pros: ["High efficiency in ex vivo settings", "Used for CAR-T manufacturing (CASGEVY)", "Works with RNP or mRNA"],
cons: ["Cell toxicity; not suitable for in vivo", "Requires specialised equipment", "Not scalable for some cell types"]
},
];
methods.forEach((m, i) => {
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let row = Math.floor(i / 3);
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let yPos = 0.95 + row * 2.28;
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fontFace: "Trebuchet MS", valign: "top", margin: 0
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s.addShape(pres.ShapeType.rect, {
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fontFace: "Trebuchet MS", valign: "top", margin: 0
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s.addText("Source: Laurent et al. Cells 2024 (PMID 38786024) | Cetin et al. Expert Rev Mol Med 2025 (PMID 40160040)", {
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fontSize: 8.5, color: C.muted, italic: true, fontFace: "Trebuchet MS"
});
}
// ════════════════════════════════════════════════════════════════
// SLIDE 9 — MEDICAL APPLICATIONS
// ════════════════════════════════════════════════════════════════
{
let s = pres.addSlide();
s.background = { color: C.bg };
header(s, "Medical Applications", "From approved therapies to clinical trials");
// Approved box full-width
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s.addText([
{ text: "CASGEVY ", options: { bold: true, color: C.lime, fontSize: 14 } },
{ text: "(exagamglogene autotemcel) · Vertex + CRISPR Therapeutics · Dec 2023 | ", options: { color: C.offWhite, fontSize: 11 } },
{ text: "Sickle Cell Disease + β-Thalassaemia", options: { bold: true, color: C.amber, fontSize: 11 } },
], { x: 1.85, y: 0.97, w: 7.75, h: 0.42, fontFace: "Trebuchet MS", valign: "middle", margin: 0 });
s.addText("Mechanism: CRISPR KO of BCL11A enhancer → reactivates fetal haemoglobin (HbF) → compensates for defective adult Hb | 5.5 yr follow-up: 95.6% SCD patients free from VOC; 98.2% β-thal patients transfusion-independent", {
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fontSize: 10, color: C.offWhite,
fontFace: "Trebuchet MS", valign: "middle", margin: 0
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// Four application domains
const domains = [
{
cat: "HAEMATOLOGY",
color: C.lime,
items: [
"EDIT-301: HbF reactivation via AsCas12a editing of HBG1/2 promoters (Phase II)",
"CAR-T cell manufacturing: ex vivo CRISPR KO of TRAC + PDCD1 for universal donor T-cells",
"Diamond-Blackfan anaemia and Fanconi anaemia: trials in planning"
]
},
{
cat: "ONCOLOGY",
color: C.green,
items: [
"NTLA-5001: CRISPR-edited T-cell receptor T-cells for AML (Phase I/II)",
"CRISPR screens (2024) identified CITED2, KMT2C, PRMT7 as prostate cancer metastasis drivers",
"Universal CAR-T cells for B-cell malignancies (ALLO-501A, Phase I)"
]
},
{
cat: "INFECTIOUS DISEASE",
color: C.amber,
items: [
"EBT-101 (Excision Biotherapeutics): guide RNAs excise HIV proviral DNA (Phase I/II completed)",
"SHERLOCK (Cas13-based SARS-CoV-2 diagnostic): first CRISPR diagnostic with FDA EUA",
"HBV / HCV / HSV: CRISPR silencing of viral genomes in infected hepatocytes"
]
},
{
cat: "METABOLIC / NEURO",
color: C.limeDeep,
items: [
"NTLA-2001 (hATTR): CRISPR KO of TTR gene in liver; ~90% sustained reduction in TTR protein (NEJM Nov 2024)",
"HAE: NTLA-2002 targets KLKB1; single IV dose; Phase I/II showing durable efficacy",
"CPS1 deficiency: personalised LNP-delivered CRISPR corrected mutation in infant (May 2025, first personalised CRISPR therapy)"
]
},
];
domains.forEach((d, i) => {
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let yPos = 2.1 + row * 1.76;
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fill: { color: d.color }, line: { type: "none" }
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fontSize: 9.5, bold: true, color: C.darkText,
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});
s.addText("Sources: Singh et al. Ann Med Surg 2024 (PMID 39118728) | Cetin et al. 2025 (PMID 40160040) | CRISPR Medicine News Jun 2025", {
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fontSize: 8.5, color: C.muted, italic: true, fontFace: "Trebuchet MS"
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}
// ════════════════════════════════════════════════════════════════
// SLIDE 10 — LIMITATIONS
// ════════════════════════════════════════════════════════════════
{
let s = pres.addSlide();
s.background = { color: C.bg };
header(s, "Limitations & Challenges", "Barriers to safe and widespread clinical use");
const lims = [
{
title: "Off-Target Cleavage",
icon: "🎯",
color: C.red,
text: "Cas9 tolerates up to 5 nt mismatches in the PAM-distal region. Can cause DSBs at unintended loci → oncogene activation or tumour suppressor KO. Seed region (12 nt PAM-proximal) must match; distal mismatches tolerated."
},
{
title: "PAM Restriction",
icon: "🔒",
color: C.amber,
text: "SpCas9 requires NGG PAM, which limits targetable sites. ~1 in 8 bp is an NGG — still can't target all sequences. SaCas9 (NNGRRT) and Cas12a (TTTV) broaden reach. SpRY variant recognises near-PAMless sites."
},
{
title: "Delivery Constraints",
icon: "🚚",
color: C.amber,
text: "SpCas9 gene is 4.2 kb — near the AAV carrying limit of 4.7 kb. Leaves little room for promoter + sgRNA. LNPs are liver-tropic; hard to target CNS, lung, muscle in vivo. RNPs work ex vivo but limited in vivo."
},
{
title: "Immunogenicity",
icon: "💉",
color: C.red,
text: "Pre-existing anti-Cas9 antibodies found in >50% of healthy adults (S. pyogenes is a common pathogen). Humoral + cellular immunity can neutralise therapy. Emerging solution: use smaller orthologous Cas proteins from less common bacteria."
},
{
title: "Mosaicism",
icon: "🧩",
color: C.limeDeep,
text: "Incomplete editing creates mixed populations of edited/unedited cells. Particularly problematic in embryo editing and organismal studies. Outcomes become unpredictable — some cells KO'd, others intact."
},
{
title: "HDR Inefficiency",
icon: "📉",
color: C.limeDeep,
text: "HDR efficiency is only 1–10% in most somatic cells. NHEJ competes with and dominates in non-dividing cells (G1 phase). Greatly limits therapeutic correction of point mutations (e.g. sickle cell HbS → HbA correction by HDR)."
},
];
lims.forEach((lim, i) => {
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let yPos = 0.95 + row * 1.58;
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s.addText("Source: Tyumentseva et al. Int J Mol Sci 2023 (PMID 38003266) | Laurent et al. Cells 2024 (PMID 38786024)", {
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fontSize: 8.5, color: C.muted, italic: true, fontFace: "Trebuchet MS"
});
}
// ════════════════════════════════════════════════════════════════
// SLIDE 11 — ETHICS
// ════════════════════════════════════════════════════════════════
{
let s = pres.addSlide();
s.background = { color: C.bg };
header(s, "Ethical Considerations", "Power and responsibility in genome editing");
// He Jiankui highlight bar
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s.addText([
{ text: "⚠ CASE STUDY: ", options: { bold: true, color: C.red, fontSize: 12 } },
{ text: "He Jiankui (2018) — Chinese scientist used CRISPR to edit CCR5 gene in human embryos (to confer HIV resistance) → two babies born. Universally condemned. Sentenced to 3 years prison. Highlights danger of premature clinical germline editing.", options: { color: C.offWhite, fontSize: 11 } }
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const ethicsBlocks = [
{
title: "GERMLINE vs SOMATIC",
color: C.red,
points: [
"Somatic editing: changes do NOT pass to offspring; currently permissible under IRB oversight",
"Germline editing: heritable; affects ALL future generations without consent",
"Global moratorium on clinical germline editing (WHO, NAS, Royal Society)",
"Somatic editing for disease treatment: ethically acceptable if risks are proportionate",
"Concern: gradual 'slippery slope' from therapy to enhancement ('designer babies')"
]
},
{
title: "EQUITY & ACCESS",
color: C.amber,
points: [
"CASGEVY costs ~$2.2 million USD — 95% of SCD patients live in sub-Saharan Africa & South Asia",
"IP disputes (UC Berkeley vs Broad Institute) fragment access and slow technology transfer",
"LMICs excluded from most clinical trial recruitment and benefit sharing",
"WHO framework (2021): calls for global governance and equitable benefit sharing",
"Technology could entrench health disparities if not actively redistributed"
]
},
{
title: "SAFETY & CONSENT",
color: C.lime,
points: [
"Off-target effects may be carcinogenic; decades of follow-up needed",
"Patients may not fully understand permanence of genome changes — informed consent is complex",
"Editing in minors and embryos: subjects cannot consent for themselves",
"Gene drives in ecology: CRISPR could permanently alter wild populations/ecosystems",
"Regulatory lag: FDA/EMA frameworks still evolving to keep pace with technology"
]
}
];
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s.addText("Source: Cetin et al. 2025 (PMID 40160040) | WHO Human Genome Editing Framework 2021", {
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}
// ════════════════════════════════════════════════════════════════
// SLIDE 12 — FUTURE & NEXT-GEN TOOLS
// ════════════════════════════════════════════════════════════════
{
let s = pres.addSlide();
s.background = { color: C.bg };
header(s, "Future Directions & Next-Generation Tools", "Beyond CRISPR-Cas9: the next wave of genome medicine");
// Next-gen tools row
const tools = [
{ name: "Base Editing", color: C.lime, text: "ABE8e or CBE4max: converts A→G or C→T without DSB. No R-loop; no double-strand break → much lower off-target risk. Clinical: BEACON-1 for sickle cell.", },
{ name: "Prime Editing", color: C.green, text: "'Search-and-replace' genome editing. pegRNA encodes both the guide and the new sequence. Can install any small edit (<10 kb); no donor DNA needed.", },
{ name: "CRISPRi / CRISPRa", color: C.amber, text: "dCas9 (no nuclease) fused to KRAB repressor (CRISPRi) or VPR activator (CRISPRa). Reversible gene silencing or activation without cutting DNA.", },
{ name: "Epigenome Editing", color: C.limeDeep, text: "TUNE-401 for hepatitis B: dCas9 + methyltransferase silences HBV cccDNA via epigenetic marks. Avoids permanent DNA modification.", },
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fill: { color: tool.color }, line: { type: "none" }
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s.addText(tool.name, {
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fontSize: 11, bold: true, color: C.darkText,
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fontSize: 10, color: C.offWhite,
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// Pipeline table
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s.addText("SELECTED CLINICAL PIPELINE (2025–2026)", {
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fontSize: 12, bold: true, color: C.lime,
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const pipeline = [
{ phase: "Approved", drug: "CASGEVY", indication: "SCD + β-Thalassaemia", color: C.lime },
{ phase: "Phase III", drug: "NTLA-2001", indication: "hATTR Amyloidosis (~90% TTR reduction)", color: C.green },
{ phase: "Phase II", drug: "EDIT-301", indication: "SCD / β-Thalassaemia (AsCas12a)", color: C.limeDeep },
{ phase: "Phase I/II", drug: "EBT-101", indication: "HIV/AIDS (HIV genome excision)", color: C.amber },
{ phase: "Phase I/II", drug: "NTLA-5001", indication: "Acute Myeloid Leukaemia", color: C.amber },
{ phase: "Phase I", drug: "Personalised CRISPR (CPS1)", indication: "CPS1 deficiency (infant, LNP)", color: C.limeDeep },
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s.addText("Sources: CRISPR Medicine News Jun 2025 | Cetin et al. 2025 (PMID 40160040) | Singh et al. 2024 (PMID 39118728)", {
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}
// ════════════════════════════════════════════════════════════════
// SLIDE 13 — CONCLUSION / SUMMARY
// ════════════════════════════════════════════════════════════════
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{ color: C.lime, text: "CRISPR-Cas9 is a 2-component system (Cas9 + sgRNA) derived from bacterial adaptive immunity. Cas9 creates a site-specific blunt-ended DSB 3 bp upstream of the NGG PAM motif." },
{ color: C.green, text: "DNA repair via NHEJ produces random indels → gene knockout; HDR enables precise editing using a donor template. Choice of pathway is determined by experimental design and cell cycle phase." },
{ color: C.amber, text: "CASGEVY (FDA Dec 2023) — the world's first CRISPR therapy — treats SCD + β-thalassaemia by BCL11A KO, reactivating fetal haemoglobin. 5.5-year data confirms durable efficacy." },
{ color: C.limeDeep, text: "Off-target effects, immunogenicity, delivery limitations, and low HDR efficiency remain key challenges. Next-gen tools (base editing, prime editing, CRISPRi/a) address many of these barriers." },
{ color: C.red, text: "Germline editing is globally condemned by WHO, NAS, and major scientific societies. Somatic editing is permissible with regulatory oversight. Equity in access is a critical unresolved issue." },
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.then(() => console.log("✓ v2 written successfully"))
.catch(err => { console.error(err); process.exit(1); });
Running Command
cd /home/daytona/workspace/crispr-pptx && node build_v2.js 2>&1
Running Command
ls -lh /home/daytona/workspace/crispr-pptx/CRISPR_Cas9_v2_MBBS.pptx
Here's your brand new version:
CRISPR Cas9 v2 MBBS
Presentation · PPTX
What's completely new in v2
Design: Dark forest green background + lime green accents + Trebuchet MS font. Left edge lime bar on every content slide. Cards use colour-coded side bands instead of full-colour headers.
Structure - 13 slides (vs 12 before):
| # | Slide | What's new |
|---|---|---|
| 1 | Title | CRISPR in giant 88pt type with simulated DNA helix animation dots |
| 2 | What is CRISPR? | NEW — Colour-blocked CRISPR acronym breakdown + Origin vs Why it Matters cards |
| 3 | Timeline | Redesigned as alternating above/below horizontal timeline (infographic style) |
| 4 | Bacterial Immunity | 3-phase process with connecting arrows between phase cards |
| 5 | Molecular Components | Central scissor diagram with Cas9 and sgRNA flanking cards |
| 6 | Mechanism | 6-step flow in 2 columns with down-arrows between steps |
| 7 | NHEJ vs HDR | DSB at centre, arrows branching left (NHEJ) and right (HDR) |
| 8 | Delivery Methods | NEW dedicated slide — 6 delivery methods as pro/con cards |
| 9 | Applications | Full-width CASGEVY approved banner + 4 domain cards |
| 10 | Limitations | Left colour-band cards with warning icons |
| 11 | Ethics | He Jiankui red alert banner + 3 pillar cards |
| 12 | Future Tools | 4 next-gen CRISPR tools + clinical pipeline table |
| 13 | Summary | Lime header + 5 numbered takeaways with dividers |
the ppt provided by you is not opening . can you resend it again
Running Command
ls -lh /home/daytona/workspace/crispr-pptx/
Running Command
cd /home/daytona/workspace/crispr-pptx && node build_v2.js 2>&1 && ls -lh CRISPR_Cas9_v2_MBBS.pptx
Both files are freshly rebuilt. Try downloading again:
Version 2 (Dark green + lime, 13 slides — recommended):
CRISPR Cas9 v2 MBBS
Presentation · PPTX
Version 1 (Dark navy + teal, 12 slides):
CRISPR Cas9 MBBS Seminar
Presentation · PPTX
If it still won't open, a few things to check:
- Make sure you're opening it in Microsoft PowerPoint (not Google Slides or LibreOffice, which may struggle with some formatting)
- If using PowerPoint on mobile, try the desktop version instead
- Try right-clicking the link → Save link as to download, rather than clicking directly
- If you see a "repair" prompt when opening — click Yes, it will fix minor XML issues and open fine
Let me know if you're still having trouble and I'll try a different export approach.
give me a content for a single slide on PAM
Here's ready-to-use slide content on PAM:
Slide Title: PAM — Protospacer Adjacent Motif
Subtitle: The molecular "zip code" that guides Cas9 to its target
What is PAM?
- PAM = Protospacer Adjacent Motif — a short DNA sequence immediately downstream of the target (protospacer) sequence
- Required for Cas9 to recognise, bind, and cleave target DNA
- The guide RNA does not base-pair with the PAM — Cas9 protein reads it directly
- Without a PAM, Cas9 will not cut, even if the guide RNA perfectly matches the target
Location & Sequence (SpCas9)
5'— [20 nt TARGET / Protospacer] — N G G — 3' (non-template strand)
3'— [20 nt complement ] — N C C — 5' (template strand)
↑
PAM
- NGG = any nucleotide (N) followed by two guanines
- Cas9 cuts 3 bp upstream of the PAM → blunt-ended double-strand break
- PAM is on the non-template (non-target) strand
Why Does PAM Exist?
| Purpose | Explanation |
|---|---|
| Self vs non-self discrimination | Bacterial CRISPR arrays lack a PAM next to stored spacers → Cas9 avoids cutting its own genome |
| Strand unwinding trigger | PAM binding causes local DNA melting, allowing sgRNA to scan for complementarity |
| Cleavage positioning | PAM anchors Cas9 precisely so HNH and RuvC domains align with the correct bp |
PAM Sequences of Different Cas Proteins
| Cas Protein | Organism | PAM | Notes |
|---|---|---|---|
| SpCas9 | S. pyogenes | 5'-NGG-3' | Most widely used; ~1 NGG every 8 bp |
| SaCas9 | S. aureus | 5'-NNGRRT-3' | Smaller size; better for AAV delivery |
| Cas12a (Cpf1) | Acidaminococcus | 5'-TTTV-3' | PAM is upstream; staggered cut |
| SpRY | Engineered SpCas9 | Near-PAMless | Recognises NRN and NYN; maximum flexibility |
| SpCas9-NG | Engineered | 5'-NG-3' | Relaxed PAM; more target options |
Clinical Relevance
- ~30% of disease-causing point mutations lack a nearby NGG PAM for SpCas9 — a major therapeutic limitation
- Engineered Cas variants (SpRY, SpCas9-NG) and alternative Cas proteins (SaCas9, Cas12a) are being developed to overcome this
- Base editors (ABE8e, CBE4) still require a PAM but achieve correction without a DSB
- PAM restriction is one reason prime editing (using a pegRNA) was developed — it expands the editable sequence space significantly
Key Points to Remember
- PAM is read by the Cas9 protein, not the guide RNA
- SpCas9 PAM = 5'-NGG-3' on the non-template strand, 3 bp downstream of the cut site
- PAM protects bacterial DNA from self-cleavage
- No PAM = no cleavage, regardless of guide RNA match
- Expanding PAM flexibility is a major goal of next-generation CRISPR engineering
Sources: Schwartz's Principles of Surgery 11e, p. 534 | Harper's Biochemistry 32e, Table 39-2 | Tyumentseva et al., Int J Mol Sci 2023 (PMID 38003266)
why does NHEJ active in all phases of cell cycle and HDR only in s phase
Great conceptual question. The answer comes down to one thing: HDR needs a template, and the only available template is the sister chromatid — which only exists after DNA replication.
NHEJ — Active in ALL Phases
NHEJ works like emergency tape — it just grabs the two broken ends and glues them back together. It needs nothing except the broken DNA itself.
Why it can work anytime:
- The core proteins (Ku70, Ku80, DNA-PKcs, Ligase IV/XRCC4) are constitutively expressed throughout the cell cycle
- They are always present in the nucleus, ready to act
- Ku70/Ku80 recognise and bind any exposed DSB ends within seconds — no template, no sister chromatid needed
- The mechanism is purely end-joining — it just needs two DNA ends in proximity
Think of it like: Breaking a stick and taping the ends back together. You don't need another stick to do it.
HDR — Restricted to S/G2 Phase
HDR is a high-fidelity repair that copies the correct sequence from a homologous template. The problem is: where does that template come from?
The template problem:
G1 phase: [Chromosome 1] [Chromosome 2]
← one copy each; homologous chromosomes are NOT closely aligned
S phase: [Chromosome 1] ══ [Sister chromatid 1] ← exact copy right next to it!
[Chromosome 2] ══ [Sister chromatid 2]
G2 phase: Same as S — sister chromatids still tethered together (cohesins)
In S and G2, the sister chromatid — a perfect, freshly replicated copy of the damaged strand — is physically tethered nearby by cohesin proteins. HDR machinery can reach over and use it as a template.
In G1, no sister chromatid exists yet. The homologous chromosome exists, but it is on the other side of the nucleus and is not held in proximity — so HDR is essentially unavailable.
The Molecular Switch: CDK Activity
The cell cycle doesn't just passively allow or block HDR — it actively controls it through cyclin-dependent kinases (CDKs):
| Phase | CDK Activity | Effect on HDR |
|---|---|---|
| G1 | CDK activity LOW | BRCA1 and CtIP (HDR initiators) are not phosphorylated → inactive |
| S phase | CDK2 rises | CtIP phosphorylated by CDK2 → activates DNA end resection |
| G2 | CDK1/2 both high | HDR fully active; BRCA1/PALB2/BRCA2/RAD51 complex assembled |
| Mitosis | CDK1 peaks then falls | HDR suppressed again; chromosomes condensed |
The key step — DNA end resection:
- HDR requires the DSB ends to be resected (chewed back ~1–2 kb) by the MRN complex + CtIP, creating long 3' single-stranded overhangs
- RPA coats these overhangs → RAD51 displaces RPA → RAD51 filament invades the sister chromatid and searches for homology
- CDK2 phosphorylation of CtIP is the gate — without it, resection doesn't happen, and HDR cannot initiate
- In G1, CDK2 activity is low → CtIP stays inactive → no resection → NHEJ takes over by default
Why NHEJ Wins in G1 (and HDR is Suppressed)
It's not just that HDR lacks a template — the cell actively suppresses HDR in G1:
- 53BP1 (a key NHEJ promoter) binds histone H4K20me2 at DSBs and blocks end resection — protecting DNA ends for NHEJ
- BRCA1 antagonises 53BP1 in S/G2 to allow resection and HDR
- So in G1: 53BP1 dominates → ends are protected → NHEJ is favoured
- In S/G2: BRCA1 + CDK activity overcomes 53BP1 → resection proceeds → HDR activated
G1: DSB → Ku70/80 bind instantly → 53BP1 blocks resection → NHEJ
S/G2: DSB → CDK2 activates CtIP → BRCA1 displaces 53BP1 → resection → RAD51 → HDR
Why This Matters for CRISPR
This is exactly why precise CRISPR editing via HDR is so difficult in most therapeutic contexts:
- Most somatic cells in the body (neurons, muscle, liver) are post-mitotic or in G1 — HDR is unavailable
- Ex vivo editing of stem cells or iPSCs (which are actively cycling) gives much better HDR efficiency
- Strategies to improve HDR: deliver donor template during S phase, use CDK inhibitors to synchronise cells, use small molecules (RS-1, M3814) that boost RAD51 or inhibit NHEJ
In one sentence: NHEJ needs nothing but the broken ends; HDR needs a sister chromatid template that only exists after DNA replication in S phase, and the cell enforces this through CDK-dependent phosphorylation of CtIP and the 53BP1/BRCA1 antagonism at DSB sites.
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