Goal: 20 nt chunna jo cut ko aapke gene of interest tak direct kare, maximum on-target aur minimum off-target activity ke saath.
Apna target region dhundho genomic DNA mein (jaise woh codon jise aap knock out karna chahte ho).
PAM (NGG) scan karo us region ke paas kisi bhi strand par.
Us PAM ke immediately 5' wale 20 nt lo DNA par, yahi protospacer hai.
Spacer likhna = protospacer strand jaisa hi sequence (yaani identical to that strand jo guide se pair nahi karta), taaki spacer target (bottom) strand ka complementary ho. Cas9 PAM ke ~3 bp upstream (5') par kaatta hai.
Specificity score karo: 20-mer ko BLAST karo poore genome ke khilaaf. Un guides ko reject karo jinke close matches kahin aur hain (khaskar PAM-proximal "seed" region mein matches, PAM ke nearest ~10–12 nt, jo targeting mein dominant role play karte hain).
On-target efficiency check karo: extreme GC content se bachao (aim ~40–60%), poly-T stretches se bachao (TTTT transcription ko U6/Pol III promoter se terminate karta hai), aur aksar 5' end par G prefer karo U6 promoter ke liye.
CRISPR system ka kaunsa part target specificity provide karta hai?
Guide RNA (uska 20-nt spacer), target DNA ke saath base-pairing karke.
sgRNA ka spacer kya hota hai?
~20-nt ka designed sequence jo target DNA strand ka complementary hota hai.
Scaffold kya hai aur kya aap ise change karte ho?
Fixed crRNA:tracrRNA hairpin jise Cas9 bind karta hai; aap ise change NAHIN karte.
SpCas9 ke liye PAM kya hai?
5'-NGG-3', jo 20-nt protospacer ke immediately 3' par, DNA par hota hai.
Kya PAM guide RNA ka part hai?
Nahin — PAM sirf DNA par hota hai; guide na ise contain karta hai, na pair karta hai.
SpCas9 PAM ke relative kahaan kaatta hai?
NGG ke ~3 bp upstream (5') par, ek blunt double-strand break banata hai.
Specificity ke liye guide ka kaunsa region sabse zyada matter karta hai?
Seed region — PAM ke sabse nearest ~10–12 nt.
Bacteria apne stored spacers kyun nahi kaatते?
Unke CRISPR array mein adjacent PAM nahi hota, isliye Cas9 use ignore karta hai.
Guide ke liye acha GC-content range kya hai?
Roughly 40–60%; extremes aur poly-T se bachao (jo Pol III transcription terminate karta hai).
Agar single NGG bottom strand par ho, toh guide kaise design karte hain?
Bottom-strand NGG ke 5' ke 20 nt padho; spacer = woh bottom-strand sequence (T ke liye U).
Recall Feynman: ek 12-saal ke bacche ko samjhao
Socho ek robot hai jiske paas scissors hain jo ek giant book (tumhara DNA) kaat sakti hai — lekin woh ankhon par patti bandhe hai. Tum use ek sticky note dete ho jisme ek sentence likha hai (guide RNA). Robot slide karta rehta hai jab tak use ek aisi page nahi milti jiske words sticky note se match karein, phir wahan kaat deta hai. Ek catch hai: robot sirf tab kaatta hai jab ek special chhota tag "GG" sentence ke bilkul paas print ho (PAM) — isi se use pata chalta hai yeh uski apni instructions nahi hain. Toh tumhara poora kaam yeh hai: ek 20-letter sentence likho jo exactly ek jagah appear ho, ek GG tag ke bilkul paas. Sahi likha toh ek jagah kaat-ta hai; laparwahi se likha toh kai galat jagah kaatta hai.
