6.2.13 · HinglishGenetic Engineering & CRISPR
Explain base editing and prime editing
6.2.13· Biology › Genetic Engineering & CRISPR
WHY do we need them?
Base Editing
The two flavours
HOW it works (step by step)
- Guide RNA, Cas9-deaminase complex ko target tak le jaata hai.
- Cas9 DNA ko unwind karta hai, ek chhoti single-stranded loop expose karta hai jise R-loop kehte hain (non-target strand).
- Deaminase exposed window mein ek base par act karta hai (~ protospacer ke positions 4–8).
- CBE ki chemistry: deaminase cytosine se ek amino group remove karta hai → uracil (U). Cell U ko T ki tarah read karta hai.
- ABE ki chemistry: deaminase adenine ko inosine (I) mein convert karta hai. Cell I ko G ki tarah read karta hai.
- Ek nickase opposite (unedited) strand ko cut karta hai. Cell, nick dekhkar, edited strand ko template ki tarah use karta hai doosre strand ko "repair" karne ke liye — edit ko lock in karta hai.
Worked example Worked example: ABE se ek disease mutation fix karna
Maan lo ek pathogenic mutation ne coding strand par G→A change create kiya, toh yeh A read karta hai jahan healthy DNA mein G hona chahiye.
- ABE kyun use karein? Hume
Ako waapasGmein badalna hai. ABE exactly A→G karta hai. - Guide aise design karo ki mutant
Aediting window (positions 4–8) mein aa jaye. - Window kyun matter karti hai? Deaminase sirf exposed R-loop mein bases tak pahunch sakta hai; window ke bahar koi edit nahi hoti.
- Result: target strand par
A→G, doosre strand par nick, cell fix ko copy karta hai. Disease letter correct ho gaya — no DSB.
Limitation of base editing
Prime Editing
The pegRNA — the clever part
HOW it works (step by step)
- pegRNA site ko target karta hai; Cas9 ek strand ko nick karta hai (single cut, double nahi).
- Nicked DNA ka 3′ end pegRNA par PBS se pair karta hai.
- Reverse transcriptase edit ke saath naya DNA synthesize karne ke liye RT template use karta hai, directly nicked strand par.
- Ab naye (edited) DNA ka ek flap purane flap se compete karta hai. Cell ise resolve karta hai, edited flap ko rakh leta hai.
- Doosre strand par ek doosra nick (ek extra "nicking sgRNA" se guided) cell ko encourage karta hai ki doosra strand bhi edit se match kare.
Worked example Worked example: prime editing se 3 bases insert karna
Goal: ek gene mein CTG insert karna (base editors yeh nahi kar sakte).
- Prime editing kyun? Sirf prime editing bases add/remove kar sakta hai.
- pegRNA design karo jiska RT template original sequence plus
CTGencode kare. - RT template mein kyun daalna? Kyunki RT jo bhi copy karta hai woh naya DNA ban jaata hai — toh hum simply extra letters wahan "likhte" hain.
- Nick → prime → RT template copy karta hai (insert ke saath) → flap resolution →
CTGinsert ho gaya. No DSB, no donor.
Worked example Worked example: ek transversion C→G
Base editors C→G nahi kar sakte. Prime editing kar sakta hai.
- Kyun? Prime editing deamination chemistry par depend nahi karta; yeh physically strand ko RT template se rewrite karta hai.
- RT template mein jahan
Ctha wahanGencode karo → ho gaya.
Comparison
| Feature | Cas9 (classic) | Base Editing | Prime Editing |
|---|---|---|---|
| Double-strand break? | Yes | No (nick only) | No (nick only) |
| Needs donor DNA? | Yes (for HDR) | No | No |
| Point substitutions | via HDR (inefficient) | only transitions | any substitution |
| Insertions/deletions | random indels | No | Yes (small) |
| Key enzyme added | — | deaminase | reverse transcriptase |
Common Mistakes (Steel-man + Fix)
Flashcards
Base & prime editing classic Cas9 se zyada safe kyun hain?
Yeh double-strand breaks avoid karte hain (nick ya dead Cas9 use karte hain), indels/translocations kam karte hain.
Cytosine Base Editor (CBE) kaunsa conversion perform karta hai?
C→T (aur complementary strand par G→A), cytidine deamination ke through.
Adenine Base Editor (ABE) kaunsa conversion perform karta hai?
A→G (aur complementary strand par T→C), adenosine deamination ke through.
CBE kaunsa chemical intermediate banata hai, aur ise kaise read kiya jaata hai?
Uracil (U), cell dwara thymine (T) ki tarah read kiya jaata hai.
ABE kaunsa chemical intermediate banata hai, aur ise kaise read kiya jaata hai?
Inosine (I), cell dwara guanine (G) ki tarah read kiya jaata hai.
Base editors kaunse mutation types kar sakte hain — aur NAHI kar sakte?
Transitions kar sakte hain (C↔T, A↔G); transversions ya insertions/deletions nahi kar sakte.
Prime editing define karne wale do extra enzymes/RNAs kaunse hain?
Ek reverse transcriptase (Cas9 nickase se fused) aur ek pegRNA.
pegRNA ke 3′ extension mein kaunse do functional parts hote hain?
PBS (primer binding site) aur RT template (edit carry karta hai).
Prime editing koi bhi substitution plus chhote indels kyun kar sakta hai?
Desired edit RT template (RNA) mein likhi hoti hai aur DNA mein reverse-transcribe hoti hai — koi chemistry restriction nahi.
Base editing mein "bystander editing" kya hai?
Editing window (~positions 4–8) mein doosre identical bases (jaise extra C's) ki unwanted editing.
CBE vs ABE mein deaminase kaunsi enzyme family provide karti hai?
CBE APOBEC-type cytidine deaminase use karta hai; ABE engineered TadA adenosine deaminase use karta hai.
Kya prime editing ko alag donor DNA chahiye?
Nahi — template pegRNA ke andar hota hai aur reverse transcriptase dwara copy kiya jaata hai.
Recall Feynman: ek 12 saal ke bachche ko explain karo
Purana CRISPR ek typo fix karne ke liye poora page phaadne jaisa hai — bahut messy. Base editing ek magic eraser ki tarah hai jo ek letter badal deta hai (sirf kuch specific swaps, jaise 'a' ko 'g' mein badalna). Prime editing aur bhi smart hai: yeh ek chhoti sticky note saath laata hai sahi spelling ke saath, ise kitaab se press karta hai, aur ek chhoti copying machine naye letters likhti hai — toh tum koi bhi typo fix kar sakte ho ya missing letters bhi add kar sakte ho, bina page phaade.
Connections
- CRISPR-Cas9 mechanism — targeting backbone aur nickase provide karta hai jo yahan use hoti hai.
- Homology-Directed Repair (HDR) — purana, DSB-dependent precise-editing route jise yeh tools replace karte hain.
- Reverse Transcriptase — woh enzyme jo prime editing ko RNA info ko DNA mein likhne deti hai.
- Deamination of bases — base editors ke peeche chemistry (C→U, A→I).
- Point mutations & genetic disease — ek single-letter correction clinically kyun matter karta hai.
- Guide RNA design — pegRNA design tak extend hota hai (PBS + RT template).