CRISPR Study Guide

📖 Core Concepts CRISPR‑Cas system – Adaptive immune system in bacteria/archaea that captures fragments of invading DNA/RNA (spacers) and uses RNA‑guided nucleases to destroy future infections. Spacer – Short (30 bp) DNA fragment derived from a virus/plasmid, inserted into the CRISPR array; provides sequence memory. Repeat – Conserved 28‑37 bp palindromic sequence flanking each spacer; folds into a stem‑loop in the transcribed RNA. Guide RNA (gRNA) – crRNA (and sometimes tracrRNA) that base‑pairs with the target and directs a Cas nuclease. Protospacer Adjacent Motif (PAM) – Short 2‑5 bp motif next to the protospacer; required for acquisition and interference in most Type I/II systems. Class 1 vs Class 2 – Class 1 uses multi‑protein effector complexes (e.g., Cascade); Class 2 relies on a single large Cas protein (e.g., Cas9, Cas12a, Cas13). DNA vs RNA targeting – Cas9 & Cas12a cut DNA; Cas13 cuts single‑stranded RNA (and shows collateral RNase activity). 📌 Must Remember Cas9 PAM (S. pyogenes): 5′‑NGG‑3′. Cas12a PAM: 5′‑TTTV‑3′ (V = A, C, or G). Cas9 cleavage: HNH cuts the complementary strand; RuvC cuts the non‑complementary strand → blunt ends. Cas12a cleavage: Generates 5′ overhangs (staggered cuts). Cas13 collateral activity – Diagnostic platforms (SHERLOCK) exploit this non‑specific RNA cleavage after target binding. Spacer acquisition requires Cas1‑Cas2 (and often Cas4) and occurs adjacent to a PAM in Types I/II. Self‑avoidance – crRNA repeat‑derived handle prevents binding to the CRISPR locus itself; seed region must perfectly match the target. Adaptation vs Interference – Adaptation (new spacer integration) → Expression (crRNA processing) → Interference (target destruction). 🔄 Key Processes Adaptation (Spacer Acquisition) Cas1 (metal‑dependent nuclease) + Cas2 (scaffold) bind a protospacer DNA fragment. Cas4 (when present) trims to the correct length and orientation. Integration occurs at the leader‑proximal end of the array, preserving chronological order. crRNA Biogenesis Transcription of the entire CRISPR array → long pre‑crRNA. Type I/III: Cas6 (or Cas6e/f) cleaves at repeat stem‑loops. Type II: RNase III + tracrRNA processes the precursor. Mature crRNA = spacer + partial repeat handle(s). Interference DNA‑targeting (Cas9, Cas12a, Type I Cascade‑Cas3): gRNA‑Cas complex scans for complementarity + correct PAM. Binding → conformational change → nuclease activation → DNA cleavage. RNA‑targeting (Cas13): gRNA‑Cas13 binds target ssRNA → RNase domains cleave target and trigger collateral cleavage. Repair (Gene‑editing context) Double‑strand break repaired by NHEJ (indels) or HDR (templated insertion). 🔍 Key Comparisons Cas9 vs Cas12a PAM: NGG (G‑rich) vs TTTV (T‑rich). Guide RNA: crRNA + tracrRNA (or sgRNA) vs single crRNA only. Cut: blunt ends vs staggered 5′ overhangs. Class 1 (Cascade‑Cas3) vs Class 2 (Cas9) Multi‑protein surveillance complex vs single‑protein effector. Requires helicase‑nuclease (Cas3) for DNA degradation vs direct nuclease activity in Cas9. Cas13 vs DNA nucleases Targets RNA, not DNA. Shows collateral RNase activity (useful for diagnostics). ⚠️ Common Misunderstandings “Any PAM works for any Cas.” – PAM specificity is strict: Cas9 needs NGG; Cas12a needs TTTV; wrong PAM = no cleavage. “Cas9 cuts both strands at the same site.” – HNH and RuvC domains cut opposite strands at slightly offset positions, producing a blunt end only after alignment. “CRISPR always creates double‑strand breaks.” – Cas13 only cuts RNA; base editors and prime editors can modify DNA without DSBs. “Spacer acquisition is random.” – It preferentially occurs next to a PAM and follows a ruler‑like mechanism for length/orientation. 🧠 Mental Models / Intuition “Lock‑and‑key + alarm” – The PAM is the lock that lets the Cas‑gRNA complex (key) open the door; without the lock, the key can’t bind. “Chronological diary” – New spacers are added at the front (leader‑proximal), so the CRISPR array reads like a diary with the most recent infection first. “One‑off vs collateral” – Cas9/Cas12a are precise assassins (single target); Cas13 is a fire alarm that, once triggered, burns everything nearby (collateral RNase). 🚩 Exceptions & Edge Cases Type III systems – Do not require a PAM for acquisition or interference; instead they rely on transcription‑dependent targeting. Primed acquisition – A partially matching spacer can boost uptake of additional spacers from the same invader, even if the original match is imperfect. Self‑targeting avoidance – Mismatches in the repeat‑derived handle (outside the spacer) prevent Cas from cleaving its own CRISPR locus. 📍 When to Use Which Need blunt DSB → Choose Cas9 (NGG PAM). Prefer staggered cuts for seamless insertions → Use Cas12a (TTTV PAM). Target RNA or develop a diagnostic assay → Deploy Cas13 (collateral cleavage). Require high specificity → Use engineered high‑fidelity Cas9 variants or Cas12a (intrinsically more specific due to longer PAM). Multiplex editing in bacteria → Class 1 Cascade‑Cas3 can process many crRNAs simultaneously. 👀 Patterns to Recognize PAM + seed match → Correct target (look for NGG/TTTV immediately downstream of a perfect 8‑10 nt seed). Staggered vs blunt pattern – In gel images, Cas12a cuts produce 5′ overhangs visible as offset bands; Cas9 gives symmetrical blunt ends. Collateral signal – In SHERLOCK/DETECTR assays, a rapid fluorescence rise indicates Cas13/Cas12a activation after target detection. 🗂️ Exam Traps “Cas9 works with any PAM” – Wrong; only NGG (or engineered variants). “All CRISPR types need tracrRNA – Only Type II (Cas9) requires tracrRNA; Cas12a and many Class 1 systems do not. “Spacer acquisition is independent of PAM – Incorrect for Types I/II; PAM is essential for selecting protospacers. “Cas13 can edit DNA – Misleading; it only cleaves RNA. “Cas3 is a nuclease that cuts both strands directly – Actually a helicase‑nuclease that unwinds DNA and degrades the displaced strand, not a simple endonuclease. --- Study tip: Memorize the PAMs and the type of cut (blunt vs staggered) for each major nuclease. Then, for any question, locate the PAM, check seed complementarity, and decide which Cas protein fits the scenario. Good luck!