{"format":"molecular-machines.cards","version":1,"updated":"2026-10-01","decks":[{"machine":"cas9","name":"Cas9","count":28,"url":"/learn/cards/cas9.json"}],"cards":[{"id":"cas9-job","machine":"cas9","kind":"qa","prompt":"What does Cas9 do for the bacterium that carries it?","answer":"It destroys invading DNA.","explanation":"Cas9 is a bacterial immune protein. It cuts the DNA of an invader, so the cell survives the attack.","section":"summary","topic":"purpose","sources":["machine:summary","machine:tagline"],"tags":["immunity","dna"],"difficulty":1,"url":"/machines/cas9#summary","cites":[{"source":"machine:summary","machine":"cas9","label":"Summary","section":"summary"},{"source":"machine:tagline","machine":"cas9","label":"Summary","section":"summary"}]},{"id":"cas9-address-in-rna","machine":"cas9","kind":"qa","prompt":"In Cas9, what part of the machine holds the address of the target?","answer":"The guide RNA, not the protein.","explanation":"Because the address sits in the RNA, one enzyme reaches any sequence: a new guide gives a new target.","section":"summary","topic":"purpose","sources":["machine:summary"],"tags":["guide-rna","targeting"],"difficulty":1,"url":"/machines/cas9#summary","cites":[{"source":"machine:summary","machine":"cas9","label":"Summary","section":"summary"}]},{"id":"cas9-energy","machine":"cas9","kind":"cloze","prompt":"Cas9 spends no ATP on targeting; cleavage needs {{Mg2+}} and the free energy of RNA-DNA base pairing.","answer":"Mg2+","explanation":"Nothing in the search or the cut is paid for with ATP. The metal serves the two nuclease sites.","section":"summary","topic":"purpose","sources":["machine:energy"],"tags":["energy","magnesium"],"difficulty":2,"url":"/machines/cas9#summary","cites":[{"source":"machine:energy","machine":"cas9","label":"Summary","section":"summary"}]},{"id":"cas9-hnh-strand","machine":"cas9","kind":"qa","prompt":"Which DNA strand does the HNH domain of Cas9 cut?","answer":"The target strand, the one that base pairs with the guide.","explanation":"HNH, residues 770-921, carries His840 as its proton acceptor. Its position, not its chemistry, decides whether cleavage happens.","section":"summary","topic":"parts","sources":["component:HNH","stat:Nuclease domains","ref:jinek2012"],"tags":["hnh","domains"],"difficulty":1,"url":"/machines/cas9#summary","cites":[{"source":"component:HNH","machine":"cas9","label":"Part: HNH","section":"summary"},{"source":"stat:Nuclease domains","machine":"cas9","label":"Key number: Nuclease domains","section":"summary"},{"source":"ref:jinek2012","machine":"cas9","label":"Jinek et al.","section":"sources","anchor":"ref-jinek2012","href":"https://doi.org/10.1126/science.1225829"}]},{"id":"cas9-ruvc-strand","machine":"cas9","kind":"qa","prompt":"Which DNA strand does the RuvC domain of Cas9 cut?","answer":"The non-target strand, the displaced strand that carries the PAM.","explanation":"RuvC is built from three segments of the chain (1-62, 718-765 and 925-1102) and uses Asp10 as its catalytic residue.","section":"summary","topic":"parts","sources":["component:RuvC","component:non-target DNA strand","ref:jinek2012"],"tags":["ruvc","domains"],"difficulty":2,"url":"/machines/cas9#summary","cites":[{"source":"component:RuvC","machine":"cas9","label":"Part: RuvC","section":"summary"},{"source":"component:non-target DNA strand","machine":"cas9","label":"Part: non-target DNA strand","section":"summary"},{"source":"ref:jinek2012","machine":"cas9","label":"Jinek et al.","section":"sources","anchor":"ref-jinek2012","href":"https://doi.org/10.1126/science.1225829"}]},{"id":"cas9-catalytic-residues","machine":"cas9","kind":"cloze","prompt":"The two catalytic residues of SpCas9 are {{D10 and H840}}.","answer":"D10 and H840","explanation":"Asp10 serves the RuvC-like site and His840 is the proton acceptor of the HNH site, so changing both removes all cutting.","section":"summary","topic":"numbers","sources":["stat:Catalytic residues","ref:uniprot_q99zw2"],"tags":["active-site","residues"],"difficulty":2,"url":"/machines/cas9#summary","cites":[{"source":"stat:Catalytic residues","machine":"cas9","label":"Key number: Catalytic residues","section":"summary"},{"source":"ref:uniprot_q99zw2","machine":"cas9","label":"UniProt Q99ZW2 (CAS9_STRP1), Streptococcus pyogenes serotype M1","section":"sources","anchor":"ref-uniprot_q99zw2","href":"https://rest.uniprot.org/uniprotkb/Q99ZW2.json"}]},{"id":"cas9-pam-arginines","machine":"cas9","kind":"qa","prompt":"Which two Cas9 residues read the two guanines of the NGG PAM?","answer":"Arg1333 and Arg1335.","explanation":"The PAM is the protospacer adjacent motif. Reading it limits which sites the enzyme can reach.","section":"summary","topic":"parts","sources":["stat:PAM required","fact:s-pam","ref:anders2014"],"tags":["pam","residues"],"difficulty":2,"url":"/machines/cas9#summary","cites":[{"source":"stat:PAM required","machine":"cas9","label":"Key number: PAM required","section":"summary"},{"source":"fact:s-pam","machine":"cas9","label":"Arginines that read the PAM: 2 arginines","section":"story"},{"source":"ref:anders2014","machine":"cas9","label":"Anders et al.","section":"sources","anchor":"ref-anders2014","href":"https://doi.org/10.1038/nature13579"}]},{"id":"cas9-prime-editing-scope","machine":"cas9","kind":"qa","prompt":"What share of known disease-associated genetic variants do the prime editing authors estimate it could in principle correct?","answer":"Up to 89 percent.","explanation":"Prime editing installs insertions, deletions and every point mutation class without double-strand breaks. The 89 percent is an author estimate of scope, not a measured correction rate.","section":"summary","topic":"debate","sources":["frontier:Writing without breaks","evidence:prime-editing-scope","ref:anzalone2019"],"tags":["prime-editing","frontier"],"difficulty":2,"url":"/machines/cas9#summary","cites":[{"source":"frontier:Writing without breaks","machine":"cas9","label":"Open question: Writing without breaks","section":"summary"},{"source":"evidence:prime-editing-scope","machine":"cas9","label":"Share of known pathogenic human variants that prime editing could in principle correct (Anzalone AV 2019)","section":"evidence","anchor":"ev-prime-editing-scope"},{"source":"ref:anzalone2019","machine":"cas9","label":"Anzalone et al.","section":"sources","anchor":"ref-anzalone2019","href":"https://doi.org/10.1038/s41586-019-1711-4"}]},{"id":"cas9-open-frontier","machine":"cas9","kind":"qa","prompt":"Which goal on the Cas9 frontier list is still only proposed rather than demonstrated?","answer":"Reliable in-place writing of whole genes or chromosomes.","explanation":"Today's editors change a few bases or insert short cassettes. Programmable writing of long sequences is still ahead.","section":"summary","topic":"debate","sources":["frontier:Programmable writing of long sequences"],"tags":["frontier","editing"],"difficulty":2,"url":"/machines/cas9#summary","cites":[{"source":"frontier:Programmable writing of long sequences","machine":"cas9","label":"Open question: Programmable writing of long sequences","section":"summary"}]},{"id":"cas9-guide-arms-protein","machine":"cas9","kind":"qa","prompt":"What has to happen before Cas9 can accept DNA at all?","answer":"The guide RNA must bind and turn the two lobes toward each other, forming the central channel.","explanation":"Apo Cas9 has no channel for DNA. Most of the rearrangement happens before any DNA arrives.","section":"mechanism","topic":"cycle","sources":["mechanism:The guide RNA arms the protein","ref:jinek2014"],"tags":["guide-rna","conformation"],"difficulty":2,"url":"/machines/cas9#mechanism","cites":[{"source":"mechanism:The guide RNA arms the protein","machine":"cas9","label":"Step: The guide RNA arms the protein","section":"mechanism"},{"source":"ref:jinek2014","machine":"cas9","label":"Jinek et al.","section":"sources","anchor":"ref-jinek2014","href":"https://doi.org/10.1126/science.1247997"}]},{"id":"cas9-skips-matches-without-pam","machine":"cas9","kind":"qa","prompt":"Why does Cas9 pass over a perfect sequence match that has no nearby PAM?","answer":"Binding and cleavage both need a short PAM next to the target.","explanation":"During the search, affinity for non-target DNA scales with PAM density, so the PAM is what the enzyme checks first.","section":"mechanism","topic":"cycle","sources":["mechanism:Cas9 reads PAMs while it scans","ref:sternberg2014"],"tags":["pam","search"],"difficulty":2,"url":"/machines/cas9#mechanism","cites":[{"source":"mechanism:Cas9 reads PAMs while it scans","machine":"cas9","label":"Step: Cas9 reads PAMs while it scans","section":"mechanism"},{"source":"ref:sternberg2014","machine":"cas9","label":"Sternberg et al.","section":"sources","anchor":"ref-sternberg2014","href":"https://doi.org/10.1038/nature13011"}]},{"id":"cas9-rloop-growth","machine":"cas9","kind":"cloze","prompt":"Strand separation in Cas9 starts at the {{PAM}}, and the RNA-DNA heteroduplex then grows away from there toward the far end of the target.","answer":"PAM","explanation":"That directional growth of base pairing builds the R-loop.","section":"mechanism","topic":"cycle","sources":["mechanism:The strands separate at the PAM","ref:jiang2016"],"tags":["r-loop","pam"],"difficulty":2,"url":"/machines/cas9#mechanism","cites":[{"source":"mechanism:The strands separate at the PAM","machine":"cas9","label":"Step: The strands separate at the PAM","section":"mechanism"},{"source":"ref:jiang2016","machine":"cas9","label":"Jiang et al.","section":"sources","anchor":"ref-jiang2016","href":"https://doi.org/10.1126/science.aad8282"}]},{"id":"cas9-first-checkpoint","machine":"cas9","kind":"qa","prompt":"What is the first checkpoint in the Cas9 cycle, the one that allows catalysis?","answer":"PAM recognition: PAM contacts trigger catalytic activity.","explanation":"Checking the PAM first means Cas9 wastes little time on sites it cannot cut.","section":"mechanism","topic":"cycle","sources":["mechanism:PAM recognition allows catalysis","ref:sternberg2014"],"tags":["pam","checkpoint"],"difficulty":2,"url":"/machines/cas9#mechanism","cites":[{"source":"mechanism:PAM recognition allows catalysis","machine":"cas9","label":"Step: PAM recognition allows catalysis","section":"mechanism"},{"source":"ref:sternberg2014","machine":"cas9","label":"Sternberg et al.","section":"sources","anchor":"ref-sternberg2014","href":"https://doi.org/10.1038/nature13011"}]},{"id":"cas9-final-checkpoint","machine":"cas9","kind":"qa","prompt":"Which step is the final specificity checkpoint of Cas9?","answer":"HNH docking into its activated conformation.","explanation":"Cleavage rate tracks how much HNH samples that conformation, and DNA binding is far less selective than cleavage.","section":"mechanism","topic":"cycle","sources":["mechanism:HNH docking decides","ref:sternberg2015"],"tags":["hnh","specificity"],"difficulty":3,"url":"/machines/cas9#mechanism","cites":[{"source":"mechanism:HNH docking decides","machine":"cas9","label":"Step: HNH docking decides","section":"mechanism"},{"source":"ref:sternberg2015","machine":"cas9","label":"Sternberg et al.","section":"sources","anchor":"ref-sternberg2015","href":"https://doi.org/10.1038/nature15544"}]},{"id":"cas9-concerted-cut","machine":"cas9","kind":"qa","prompt":"What makes the two nuclease domains of Cas9 fire together instead of one at a time?","answer":"Allosteric communication between them.","explanation":"HNH cuts the complementary strand and RuvC cuts the other, so the product is a double-strand break.","section":"mechanism","topic":"cycle","sources":["mechanism:Both strands cut together","ref:zhu2019"],"tags":["allostery","cleavage"],"difficulty":2,"url":"/machines/cas9#mechanism","cites":[{"source":"mechanism:Both strands cut together","machine":"cas9","label":"Step: Both strands cut together","section":"mechanism"},{"source":"ref:zhu2019","machine":"cas9","label":"Zhu et al.","section":"sources","anchor":"ref-zhu2019","href":"https://doi.org/10.1038/s41594-019-0258-2"}]},{"id":"cas9-ocean-infections","machine":"cas9","kind":"qa","prompt":"How many virus infections start in the ocean every second, the pressure that CRISPR immunity answers?","answer":"About 10^23 per second.","explanation":"Viruses that infect bacteria are the most numerous biological things on Earth, so bacteria have had to defend themselves for billions of years.","section":"story","topic":"numbers","sources":["fact:C2","story:summary","stop:planet","ref:suttle2007"],"tags":["phage","scale"],"difficulty":1,"url":"/machines/cas9#story","cites":[{"source":"fact:C2","machine":"cas9","label":"Virus infections in the ocean: 10^23 per second","section":"story"},{"source":"story:summary","machine":"cas9","label":"Big picture","section":"story"},{"source":"stop:planet","machine":"cas9","label":"Big picture: Planet of viruses","section":"story"},{"source":"ref:suttle2007","machine":"cas9","label":"Suttle 2007","section":"sources","href":"https://doi.org/10.1038/nrmicro1750"}]},{"id":"cas9-search-time","machine":"cas9","kind":"qa","prompt":"How long does one Cas9 need to find its target in E. coli?","answer":"About six hours.","explanation":"Measured by tracking one labelled dCas9. Because one copy is so slow, a cell keeps many copies searching.","section":"story","topic":"numbers","sources":["fact:C7","stop:search","ref:jones2017"],"tags":["search","kinetics"],"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"},{"source":"ref:jones2017","machine":"cas9","label":"Jones 2017","section":"sources","href":"https://doi.org/10.1126/science.aah7084"}]},{"id":"cas9-pam-spacing","machine":"cas9","kind":"cloze","prompt":"In random DNA an NGG PAM turns up about once every {{8}} base pairs, counting both strands.","answer":"8","explanation":"That is why the search has so many stops: about 10^6 PAM sites sit in one E. coli cell. In phage lambda DNA the spacing measured one PAM per 8.5 base pairs.","section":"story","topic":"numbers","sources":["fact:C6","ref:jones2017"],"tags":["pam","search"],"difficulty":2,"url":"/machines/cas9#story","cites":[{"source":"fact:C6","machine":"cas9","label":"Spacing of PAM sites in DNA: 8 base pairs between PAMs","section":"story"},{"source":"ref:jones2017","machine":"cas9","label":"Jones 2017","section":"sources","href":"https://doi.org/10.1126/science.aah7084"}]},{"id":"cas9-therapy-target","machine":"cas9","kind":"qa","prompt":"What does the approved Cas9 therapy cut in a patient's own blood stem cells?","answer":"The red-cell enhancer of the BCL11A gene.","explanation":"The red cells those stem cells make then produce fetal hemoglobin, which treats sickle cell disease and transfusion-dependent beta-thalassaemia.","section":"story","topic":"purpose","sources":["fact:C10","link:hemoglobin","ref:frangoul2021"],"tags":["medicine","hemoglobin"],"difficulty":2,"url":"/machines/cas9#story","cites":[{"source":"fact:C10","machine":"cas9","label":"What the approved therapy edits: BCL11A enhancer in blood stem cells","section":"story"},{"source":"link:hemoglobin","machine":"cas9","label":"Link to Hemoglobin","section":"story"},{"source":"ref:frangoul2021","machine":"cas9","label":"Frangoul 2021","section":"sources","href":"https://doi.org/10.1056/NEJMoa2031054"}]},{"id":"cas9-scissors-analogy-breaks","machine":"cas9","kind":"qa","prompt":"Calling Cas9 search-and-cut scissors gets one tool and one target right. What does it get wrong?","answer":"Scissors are aimed; Cas9 is not. It bumps into DNA at random, checks for a PAM and moves on.","explanation":"That random search is why finding one target takes hours.","section":"story","topic":"purpose","sources":["analogy:Search-and-cut scissors"],"tags":["analogy","search"],"difficulty":2,"url":"/machines/cas9#story","cites":[{"source":"analogy:Search-and-cut scissors","machine":"cas9","label":"Analogy: Search-and-cut scissors","section":"story"}]},{"id":"cas9-immune-memory-analogy-breaks","machine":"cas9","kind":"qa","prompt":"How is the bacterial CRISPR memory unlike our own immune memory?","answer":"It is written into the genome, so daughter cells inherit it.","explanation":"Both remember past infections and respond faster, but our antibody memory is not passed to our children.","section":"story","topic":"purpose","sources":["analogy:An immune memory","stop:attack"],"tags":["analogy","immunity"],"difficulty":2,"url":"/machines/cas9#story","cites":[{"source":"analogy:An immune memory","machine":"cas9","label":"Analogy: An immune memory","section":"story"},{"source":"stop:attack","machine":"cas9","label":"Big picture: Attack and memory","section":"story"}]},{"id":"cas9-residues-crystallised","machine":"cas9","kind":"cloze","prompt":"The ternary complex structure crystallised a full-length SpCas9 chain of {{1368}} residues.","answer":"1368","explanation":"X-ray crystallography at 2.5 A, with a 98-nucleotide single-guide RNA and a 23-nucleotide target DNA, anchors the sequence length in a primary structure study.","section":"evidence","topic":"numbers","sources":["evidence:spcas9-length-ternary-structure","stat:Residues","ref:nishimasu2014"],"tags":["structure","size"],"difficulty":1,"url":"/machines/cas9#evidence","cites":[{"source":"evidence:spcas9-length-ternary-structure","machine":"cas9","label":"Length of the SpCas9 polypeptide used for the ternary complex structure (Nishimasu H 2014)","section":"evidence","anchor":"ev-spcas9-length-ternary-structure"},{"source":"stat:Residues","machine":"cas9","label":"Key number: Residues","section":"summary"},{"source":"ref:nishimasu2014","machine":"cas9","label":"Nishimasu et al.","section":"sources","anchor":"ref-nishimasu2014","href":"https://doi.org/10.1016/j.cell.2014.02.001"}]},{"id":"cas9-single-mutant-nicks","machine":"cas9","kind":"qa","prompt":"What does a Cas9 carrying only one of the two active-site mutations do to DNA?","answer":"It nicks one strand instead of cutting both.","explanation":"Point mutants assayed on paired target sites offset by 4 base pairs showed which residue serves which strand: D10 for RuvC, H840 for HNH.","section":"evidence","topic":"numbers","sources":["evidence:catalytic-residues-nicking","stat:Catalytic residues"],"tags":["nickase","active-site"],"difficulty":2,"url":"/machines/cas9#evidence","cites":[{"source":"evidence:catalytic-residues-nicking","machine":"cas9","label":"Which active-site residue serves which strand (Nishimasu H 2014)","section":"evidence","anchor":"ev-catalytic-residues-nicking"},{"source":"stat:Catalytic residues","machine":"cas9","label":"Key number: Catalytic residues","section":"summary"}]},{"id":"cas9-binding-vs-cleavage-kd","machine":"cas9","kind":"qa","prompt":"Gel binding assays gave dCas9 a Kd of 0.80 nM on target. What was the Kd with 8 PAM-distal mismatches?","answer":"20 nM.","explanation":"A 25-fold spread in affinity sits beside cleavage that drops to undetectable, which is why binding is called far less selective than cleavage.","section":"evidence","topic":"numbers","sources":["evidence:binding-less-selective-than-cleavage","ref:sternberg2015"],"tags":["affinity","specificity"],"difficulty":3,"url":"/machines/cas9#evidence","cites":[{"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":"ref:sternberg2015","machine":"cas9","label":"Sternberg et al.","section":"sources","anchor":"ref-sternberg2015","href":"https://doi.org/10.1038/nature15544"}]},{"id":"cas9-hnh-fret-method","machine":"cas9","kind":"qa","prompt":"How was the link between HNH conformation and cleavage rate in Cas9 measured?","answer":"Intramolecular FRET between dyes on REC1 and HNH, compared with cleavage rate constants on the same substrates.","explanation":"With enough PAM-distal mismatches the domain stays undocked and no cleavage is detected, which makes the conformational step the checkpoint.","section":"evidence","topic":"numbers","sources":["evidence:hnh-conformation-controls-cleavage","mechanism:HNH docking decides"],"tags":["fret","method"],"difficulty":3,"url":"/machines/cas9#evidence","cites":[{"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"},{"source":"mechanism:HNH docking decides","machine":"cas9","label":"Step: HNH docking decides","section":"mechanism"}]},{"id":"cas9-hnh-swing-distance","machine":"cas9","kind":"cloze","prompt":"Cryo-EM of the active Cas9 complex with magnesium shows the HNH domain travelling about {{34}} A to reach the target-strand scissile bond.","answer":"34","explanation":"Three states were resolved at about 3.3 A: pre-catalytic, post-catalytic and product. The motion is a translation with a rotation about a central axis.","section":"evidence","topic":"numbers","sources":["evidence:hnh-swing-to-cut-site","stat:HNH approach to the cut site","ref:zhu2019"],"tags":["cryo-em","hnh"],"difficulty":2,"url":"/machines/cas9#evidence","cites":[{"source":"evidence:hnh-swing-to-cut-site","machine":"cas9","label":"Distance the HNH domain travels to reach the target-strand scissile bond (Zhu X 2019)","section":"evidence","anchor":"ev-hnh-swing-to-cut-site"},{"source":"stat:HNH approach to the cut site","machine":"cas9","label":"Key number: HNH approach to the cut site","section":"summary"},{"source":"ref:zhu2019","machine":"cas9","label":"Zhu et al.","section":"sources","anchor":"ref-zhu2019","href":"https://doi.org/10.1038/s41594-019-0258-2"}]},{"id":"cas9-cut-position","machine":"cas9","kind":"qa","prompt":"Where does Cas9 cut the target strand, relative to the PAM?","answer":"Between the third and fourth nucleotide upstream of the PAM.","explanation":"Read from cryo-EM density across the cleavage site in the pre-catalytic, post-catalytic and product states.","section":"evidence","topic":"numbers","sources":["evidence:cut-position-upstream-of-pam","ref:zhu2019"],"tags":["cleavage","cryo-em"],"difficulty":2,"url":"/machines/cas9#evidence","cites":[{"source":"evidence:cut-position-upstream-of-pam","machine":"cas9","label":"Where the target strand is cut, relative to the PAM (Zhu X 2019)","section":"evidence","anchor":"ev-cut-position-upstream-of-pam"},{"source":"ref:zhu2019","machine":"cas9","label":"Zhu et al.","section":"sources","anchor":"ref-zhu2019","href":"https://doi.org/10.1038/s41594-019-0258-2"}]},{"id":"cas9-search-mode-dispute","machine":"cas9","kind":"qa","prompt":"Single-molecule imaging on DNA curtains found Cas9 reaching targets only by three-dimensional collisions. How did later FRET work qualify that?","answer":"It saw facilitated lateral diffusion carrying Cas9 from one PAM to a neighbouring one.","explanation":"Over short distances lateral diffusion competes with three-dimensional diffusion; no rate for it is confirmed from the abstract that is available.","section":"evidence","topic":"debate","sources":["evidence:lateral-diffusion-pam-search","evidence:nonspecific-dwell-times"],"tags":["search","single-molecule"],"difficulty":3,"url":"/machines/cas9#evidence","cites":[{"source":"evidence:lateral-diffusion-pam-search","machine":"cas9","label":"How Cas9 moves between nearby PAMs while searching (Globyte V 2019)","section":"evidence","anchor":"ev-lateral-diffusion-pam-search"},{"source":"evidence:nonspecific-dwell-times","machine":"cas9","label":"Lifetime of Cas9 on non-target DNA during the search (Sternberg SH 2014)","section":"evidence","anchor":"ev-nonspecific-dwell-times"}]}]}