{"format":"molecular-machines.cards","version":1,"updated":"2026-10-12","decks":[{"machine":"cas9","name":"Cas9","count":10,"url":"/learn/cards/cas9.json"}],"cards":[{"id":"cas9-job","machine":"cas9","kind":"qa","prompt":"Viruses attack bacteria all the time, and many bacteria fight back with a memory. What does Cas9 do for the bacterium that carries it?","answer":"It finds the invader's DNA and cuts it.","section":"summary","topic":"purpose","sources":["machine:summary","story:summary"],"difficulty":1,"url":"/machines/cas9#summary","cites":[{"source":"machine:summary","machine":"cas9","label":"Summary","section":"summary"},{"source":"story:summary","machine":"cas9","label":"Big picture","section":"story"}]},{"id":"cas9-memory","machine":"cas9","kind":"qa","prompt":"When a bacterium survives a virus attack, it keeps a record of the invader in its own genome. What does it save?","answer":"A short piece of the virus's DNA, which its daughters inherit.","explanation":"The cell copies each saved piece into a guide RNA, and each guide loads into one Cas9.","section":"story","topic":"purpose","sources":["stop:attack","fact:C4","evolution:Origin in adaptive immunity"],"difficulty":1,"url":"/machines/cas9#story","cites":[{"source":"stop:attack","machine":"cas9","label":"Big picture: Attack and memory","section":"story"},{"source":"fact:C4","machine":"cas9","label":"What a bacterium gains when it survives a phage: New spacers and resistance to that phage","section":"story"},{"source":"evolution:Origin in adaptive immunity","machine":"cas9","label":"Origin in adaptive immunity","section":"summary"}]},{"id":"cas9-address-in-rna","machine":"cas9","kind":"qa","prompt":"Cas9 is one protein, but it can be aimed at almost any DNA sequence. Where does it carry the address of its target?","answer":"In its guide RNA, not in the protein.","explanation":"A new guide gives a new target. In the ribosome too, RNA carries the information.","section":"summary","topic":"parts","sources":["machine:summary","component:sgRNA"],"difficulty":1,"url":"/machines/cas9#summary","cites":[{"source":"machine:summary","machine":"cas9","label":"Summary","section":"summary"},{"source":"component:sgRNA","machine":"cas9","label":"Part: sgRNA","section":"summary"}]},{"id":"cas9-pam-search","machine":"cas9","kind":"qa","prompt":"Cas9 cannot see a matching sequence from afar. What does it check at each spot as it moves along the DNA?","answer":"A short tag next to the target, the PAM. It opens the DNA only where one fits.","explanation":"Reading this short tag first lets Cas9 skip most of the genome quickly.","section":"story","topic":"cycle","sources":["stop:search","mechanism:Cas9 reads PAMs while it scans"],"difficulty":1,"url":"/machines/cas9#story","cites":[{"source":"stop:search","machine":"cas9","label":"Big picture: The search","section":"story"},{"source":"mechanism:Cas9 reads PAMs while it scans","machine":"cas9","label":"Step: Cas9 reads PAMs while it scans","section":"mechanism"}]},{"id":"cas9-zipper","machine":"cas9","kind":"qa","prompt":"At a PAM, the guide pairs with the DNA one letter at a time, like a zipper. What happens when a wrong letter turns up early, next to the PAM?","answer":"The zipper stops and the DNA is spared.","section":"mechanism","topic":"cycle","sources":["stop:zipper","mechanism:The strands separate at the PAM","step:The first base pairs form"],"difficulty":1,"url":"/machines/cas9#mechanism","cites":[{"source":"stop:zipper","machine":"cas9","label":"Big picture: The zipper","section":"story"},{"source":"mechanism:The strands separate at the PAM","machine":"cas9","label":"Step: The strands separate at the PAM","section":"mechanism"},{"source":"step:The first base pairs form","machine":"cas9","label":"Step: The first base pairs form","section":"mechanism"}]},{"id":"cas9-double-cut","machine":"cas9","kind":"qa","prompt":"When all 20 letters of the guide match the DNA, Cas9 brings in its two cutting blades. What does it do to the DNA?","answer":"It cuts both strands, so the DNA breaks right through.","explanation":"Each blade cuts one strand, and the two fire together.","section":"mechanism","topic":"cycle","sources":["stop:machine","mechanism:Both strands cut together","fact:s-domains"],"difficulty":1,"url":"/machines/cas9#mechanism","cites":[{"source":"stop:machine","machine":"cas9","label":"Big picture: Match and cut","section":"story"},{"source":"mechanism:Both strands cut together","machine":"cas9","label":"Step: Both strands cut together","section":"mechanism"},{"source":"fact:s-domains","machine":"cas9","label":"Cutting domains: 2 HNH and RuvC","section":"story"}]},{"id":"cas9-no-fuel","machine":"cas9","kind":"qa","prompt":"Many machines in this atlas burn ATP. What powers Cas9 as it opens DNA and decides to cut?","answer":"No fuel: Cas9 spends no ATP. Base pairing between guide and DNA pays for its switch.","explanation":"Cutting also needs magnesium ions.","section":"summary","topic":"purpose","sources":["machine:energy","link:atp-synthase"],"difficulty":2,"url":"/machines/cas9#summary","cites":[{"source":"machine:energy","machine":"cas9","label":"Summary","section":"summary"},{"source":"link:atp-synthase","machine":"cas9","label":"Link to ATP synthase","section":"story"}]},{"id":"cas9-search-time","machine":"cas9","kind":"qa","prompt":"Cas9 checks spot after spot across a whole genome. Roughly how long does one Cas9 take to find its target in a bacterium?","answer":"About six hours.","explanation":"So a cell keeps many copies of Cas9.","section":"story","topic":"numbers","sources":["fact:C7","stop:search"],"difficulty":2,"url":"/machines/cas9#story","cites":[{"source":"fact:C7","machine":"cas9","label":"Time for one Cas9 to find its target in E. coli: 6 hours","section":"story"},{"source":"stop:search","machine":"cas9","label":"Big picture: The search","section":"story"}]},{"id":"cas9-cut-decision","machine":"cas9","kind":"qa","prompt":"Cas9 can stay bound to DNA that only partly matches its guide. So what makes the final call on whether it cuts?","answer":"A shape change: one cutting blade must swing into its active place, and it does so only on a full match.","explanation":"Binding is far less choosy than cutting. High-fidelity versions of Cas9 tune this switch.","section":"evidence","topic":"debate","sources":["mechanism:HNH docking decides","evidence:binding-less-selective-than-cleavage","evidence:hnh-conformation-controls-cleavage"],"difficulty":2,"url":"/machines/cas9#evidence","cites":[{"source":"mechanism:HNH docking decides","machine":"cas9","label":"Step: HNH docking decides","section":"mechanism"},{"source":"evidence:binding-less-selective-than-cleavage","machine":"cas9","label":"Binding affinity of catalytically dead Cas9 for on-target and mismatched DNA (Sternberg SH 2015)","section":"evidence","anchor":"ev-binding-less-selective-than-cleavage"},{"source":"evidence:hnh-conformation-controls-cleavage","machine":"cas9","label":"Relation between HNH domain conformation and DNA cleavage rate (Sternberg SH 2015)","section":"evidence","anchor":"ev-hnh-conformation-controls-cleavage"}]},{"id":"cas9-therapy-disease","machine":"cas9","kind":"qa","prompt":"People now use Cas9 to edit genes in patients. Which disease did the first approved Cas9 therapy treat?","answer":"Sickle cell disease (and transfusion-dependent beta-thalassaemia).","explanation":"Cas9 edits the patient's own blood stem cells, so their red cells make fetal hemoglobin.","section":"story","topic":"purpose","sources":["fact:C9","link:hemoglobin","fact:C10"],"difficulty":2,"url":"/machines/cas9#story","cites":[{"source":"fact:C9","machine":"cas9","label":"First approval of a CRISPR-Cas9 therapy: 16 Nov 2023","section":"story"},{"source":"link:hemoglobin","machine":"cas9","label":"Link to Hemoglobin","section":"story"},{"source":"fact:C10","machine":"cas9","label":"What the approved therapy edits: BCL11A enhancer in blood stem cells","section":"story"}]}]}