{ "id": "cas-nine", "title": "Cas9", "lang": "en", "source_lang": "en", "summary": "Cas9 is an RNA-guided DNA nuclease from bacterial CRISPR systems, widely used for genome editing and programmable control of gene expression.", "category": "science", "subcategory": "biology", "type": "concept", "aliases": [ "CRISPR-associated protein 9", "Cas9 nuclease", "CRISPR-associated endonuclease Cas9" ], "generated_at": "2026-10-05", "model": "pro/gpt-6.1-sol", "url": "https://aiwiki.page/en/cas-nine/", "translations": { "zh-Hans": { "url": "https://aiwiki.page/zh/cas-nine/", "json": "https://aiwiki.page/zh/cas-nine.json" } }, "sections": [ { "heading": "", "text": "Cas9 is an RNA-guided enzyme that cuts DNA at sequences specified by a guide RNA and an adjacent recognition motif. It is the principal interference protein of type II CRISPR–Cas systems, which provide sequence-specific defense against foreign genetic material in bacteria. Adapted for laboratory use, Cas9 enables genome editing by introducing targeted DNA breaks whose repair can alter a sequence. Engineered versions also serve as programmable DNA-binding proteins or components of editing systems that do not require a double-strand break. (pubmed.ncbi.nlm.nih.gov)" }, { "heading": "Biological origin and function", "text": "CRISPR systems store fragments of previously encountered foreign DNA as spacers between repeated sequences in the bacterial genome. RNA produced from these arrays supplies sequence information for recognizing corresponding genetic material, including DNA from bacteriophages and plasmids. In type II systems, Cas9 uses this information to recognize and cleave an invading DNA molecule. This constitutes a form of microbial adaptive defense, distinct from the cellular mechanisms of vertebrate immunity. (nobelprize.org)\n\nNatural Cas9 targeting depends on two RNA components:\n\n- CRISPR RNA (crRNA) contains a spacer-derived sequence complementary to the target DNA.\n- Trans-activating CRISPR RNA (tracrRNA) pairs with part of the crRNA and contributes the structure needed for association with Cas9 and DNA cleavage.\n\nFor genome engineering, these components can be fused into a single-guide RNA (sgRNA). Its targeting segment can be changed while retaining the scaffold that associates with Cas9. Thus, changing the RNA sequence can redirect the same protein to another compatible DNA site. (pubmed.ncbi.nlm.nih.gov)" }, { "heading": "Structure and target recognition", "text": "The most extensively characterized form is Cas9 from Streptococcus pyogenes, commonly called SpCas9. It contains 1,368 amino acids and has a two-lobed architecture: a recognition lobe that interacts with the guide–target complex, and a nuclease lobe containing the catalytic machinery and a region involved in recognizing the adjacent DNA motif. Structural studies have established how the guide RNA and target DNA fit within this architecture. (pmc.ncbi.nlm.nih.gov)\n\nThe protospacer adjacent motif\n\nCas9 does not recognize a target solely through RNA–DNA complementarity. It also requires an appropriate protospacer adjacent motif (PAM) beside the target sequence, or protospacer. For conventional SpCas9, the principal PAM is \\(5^{\\prime}\\text{-NGG-}3^{\\prime}\\), where \\(N\\) denotes any nucleotide. The guide typically specifies a sequence of about 20 nucleotides. The PAM is part of the DNA substrate, not part of the sequence that pairs with the guide. (nature.com)\n\nCas9–RNA complexes first interrogate DNA for compatible PAMs. PAM recognition promotes local DNA opening, allowing the guide to test the neighboring sequence. RNA–DNA pairing then extends from the PAM-proximal region. A fully complementary sequence without a suitable nearby PAM is therefore not equivalent to a functional target for conventional SpCas9. (nature.com)\n\nR-loop formation and cleavage\n\nGuide pairing creates an R-loop: an RNA–DNA hybrid accompanied by displacement of the other DNA strand. The strand complementary to the guide is conventionally called the target strand; the displaced strand is the non-target strand. Cas9 contains two catalytic domains:\n\n- The HNH domain cuts the target strand.\n- The RuvC domain cuts the non-target strand.\n\nFor SpCas9, cleavage typically occurs approximately three base pairs upstream of the PAM. Cutting both strands produces a double-strand break. Target recognition also involves conformational checkpoints: binding a DNA sequence does not automatically mean that the enzyme will complete cleavage. (pmc.ncbi.nlm.nih.gov)" }, { "heading": "Genome editing and DNA repair", "text": "Cas9 supplies a targeted lesion rather than independently writing an arbitrary replacement sequence. The resulting edit depends on DNA repair pathways, the target sequence, the cellular context, and any supplied repair template. This distinction separates targeting precision—where the enzyme acts—from editing precision—which DNA sequence remains afterward. (pmc.ncbi.nlm.nih.gov)\n\nEnd joining\n\nNon-homologous end joining and related end-joining pathways repair broken DNA ends. Repair can restore the original sequence or produce insertions and deletions, often called indels. In a coding region, some indels disrupt the reading frame or otherwise impair a gene, making Cas9 useful for gene knockout experiments. However, cleavage does not guarantee loss of gene function: different repair products can preserve activity or produce partial changes. (pmc.ncbi.nlm.nih.gov)\n\nTemplate-directed editing\n\nRepair involving homologous recombination can use a supplied DNA template to introduce a defined sequence change. This enables targeted substitutions, insertions, or integration of a sequence at a selected locus. Template-directed editing competes with other repair outcomes, so the presence of both a targeted break and a repair template does not ensure that every treated cell acquires the intended edit. (pmc.ncbi.nlm.nih.gov)\n\nMultiple guide RNAs can direct Cas9 to several sites. This permits simultaneous perturbation of different genes or removal of a region between two cleavage sites. Such multiplex targeting also expands the range of possible unintended outcomes when several DNA breaks are present together. (pmc.ncbi.nlm.nih.gov)" }, { "heading": "Natural forms and engineered variants", "text": "Cas9 denotes a family of related proteins, not a single universal sequence. Cas9 proteins from different bacteria differ in size, guide requirements, and PAM recognition. For example, Staphylococcus aureus Cas9 (SaCas9) is smaller than SpCas9 and has different targeting requirements. Its smaller coding sequence facilitated packaging of Cas9 and a guide expression cassette into a single adeno-associated viral vector in experimental work. (nature.com)\n\nSeveral engineered forms have distinct functions:\n\n- Cas9 nickases have one catalytic domain disabled and cut only one DNA strand. They can be used in paired-nick strategies or as components of other editing systems. (pmc.ncbi.nlm.nih.gov)\n- Catalytically inactive Cas9, usually called dCas9, has both nuclease activities disabled but retains RNA-directed DNA binding. It can obstruct transcription without cutting DNA. (pubmed.ncbi.nlm.nih.gov)\n- High-fidelity Cas9 variants alter interactions or conformational checkpoints to reduce cleavage of imperfectly matched targets. SpCas9-HF1 and HypaCas9 are examples. Their reported specificity improvements refer to tested guides, cell systems, and assays; they do not establish that off-target activity is impossible under all conditions. (nature.com)\n\nBase editing and prime editing\n\nBase editors combine a Cas9-derived targeting module with an enzyme that changes a DNA base chemically. Established systems include cytosine editors that enable \\(C\\!\\cdot\\!G \\rightarrow T\\!\\cdot\\!A\\) conversion and adenine editors that enable \\(A\\!\\cdot\\!T \\rightarrow G\\!\\cdot\\!C\\) conversion. These systems can introduce selected substitutions without requiring a targeted double-strand break. The chemical conversion is performed by the attached editing enzyme, not by Cas9 itself. (pubmed.ncbi.nlm.nih.gov)\n\nPrime editing combines a Cas9 nickase with a reverse transcriptase and an extended guide RNA that encodes the intended edit. The system copies information from the RNA into DNA and can introduce substitutions and small insertions or deletions without requiring a separate donor DNA template or a targeted double-strand break. Its mechanism differs from both conventional Cas9 cleavage-and-repair editing and base editing. (nature.com)" }, { "heading": "Applications", "text": "Cas9 enables experimental disruption of genes, introduction of defined genetic changes, and deletion of selected genomic regions in mammalian cells. Because targeting is specified through RNA rather than a newly designed DNA-binding protein for each locus, it also supports multiplex experiments addressing several genetic elements at once. (pmc.ncbi.nlm.nih.gov)\n\nNuclease-inactive Cas9 extends the platform beyond permanent sequence alteration. CRISPR interference (CRISPRi) uses dCas9-mediated targeting to repress gene expression, for example by obstructing transcriptional initiation or elongation. This allows researchers to investigate the effects of reduced expression without necessarily changing the underlying DNA sequence. (pubmed.ncbi.nlm.nih.gov)\n\nA clinical application is exagamglogene autotemcel, marketed as Casgevy. On December 8, 2023, it became the first therapy using CRISPR–Cas9 approved by the United States Food and Drug Administration, for eligible patients aged 12 years and older with sickle cell disease. The product contains a patient's own blood-forming stem and progenitor cells edited outside the body. Editing disrupts an erythroid-specific regulatory region of BCL11A, increasing fetal hemoglobin production rather than directly correcting the sickle-cell-associated sequence in HBB. (content.govdelivery.com)" }, { "heading": "Limitations and unintended outcomes", "text": "Off-target cleavage occurs when Cas9 acts at a site other than the intended target. Some imperfectly matched sequences can still support cleavage, so guide complementarity alone is not an absolute guarantee of specificity. Engineered high-fidelity proteins can substantially reduce this activity in tested systems, but specificity remains a property of the protein, guide, target context, and experimental conditions together. (nature.com)\n\nUnintended on-target changes are a separate issue. Even cleavage at the correct location can be followed by large deletions or complex rearrangements rather than the desired small change. These outcomes have been observed in experimental mouse and human cell systems. Consequently, examining only a short sequence surrounding the cut can miss changes extending beyond that region. (nature.com)\n\nDelivery constraints affect whether sufficient editing machinery reaches the intended cells. The size of SpCas9 is a particular limitation for some viral delivery systems; smaller Cas9 proteins can alleviate, but do not eliminate, such constraints. (nature.com)\n\nAvoiding a double-strand break does not remove every source of unintended editing. Some base editors have produced off-target RNA changes attributable to their deaminase components. Cas9-derived editing platforms therefore have to be distinguished mechanistically: the risks associated with a nuclease, a DNA-binding regulator, a base editor, and a prime editor are not identical. (nature.com)" }, { "heading": "Historical development", "text": "In 2012, Martin Jinek and colleagues demonstrated that Cas9 could be programmed for sequence-specific DNA cleavage using crRNA and tracrRNA, and that the two RNAs could be joined into an engineered single guide. This established a biochemical foundation for RNA-programmable genome editing. (pubmed.ncbi.nlm.nih.gov)\n\nIn 2013, work including that of Le Cong and colleagues demonstrated Cas9-directed genome engineering in mammalian cells. In the same year, Lei Qi and colleagues established CRISPRi using catalytically inactive Cas9, showing that programmable DNA recognition could be separated from cleavage. (pmc.ncbi.nlm.nih.gov)\n\nSubsequent developments included structural characterization of the Cas9–guide–DNA complex in 2014, cytosine base editing in 2016, adenine base editing in 2017, and prime editing in 2019. In 2020, Emmanuelle Charpentier and Jennifer A. Doudna received the Nobel Prize in Chemistry for the development of a method for genome editing. (pmc.ncbi.nlm.nih.gov)" } ], "links": [ "enzyme", "dna", "crispr", "bacteria", "genome-editing", "genome", "bacteriophage", "plasmid", "rna", "amino-acid", "nucleotide", "dna-repair", "non-homologous-end-joining", "gene", "homologous-recombination", "gene-expression", "sickle-cell-disease", "hemoglobin", "nobel-prize-in-chemistry" ], "sources": [ { "title": "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity", "url": "https://pubmed.ncbi.nlm.nih.gov/22745249/" }, { "title": "The Nobel Prize in Chemistry 2020 — Scientific Background", "url": "https://www.nobelprize.org/uploads/2020/10/advanced-chemistryprize2020.pdf" }, { "title": "Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA", "url": "https://pmc.ncbi.nlm.nih.gov/articles/4139937/" }, { "title": "DNA interrogation by the CRISPR RNA-guided endonuclease Cas9", "url": "https://www.nature.com/articles/nature13011" }, { "title": "Conformational control of DNA target cleavage by CRISPR–Cas9", "url": "https://www.nature.com/articles/nature15544" }, { "title": "Multiplex Genome Engineering Using CRISPR/Cas Systems", "url": "https://pmc.ncbi.nlm.nih.gov/articles/3795411/" }, { "title": "CRISPR/Cas9 targeting events cause complex deletions and insertions at 17 sites in the mouse genome", "url": "https://www.nature.com/articles/ncomms15464" }, { "title": "In vivo genome editing using Staphylococcus aureus Cas9", "url": "https://www.nature.com/articles/nature14299" }, { "title": "Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression", "url": "https://pubmed.ncbi.nlm.nih.gov/23452860/" }, { "title": "High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects", "url": "https://www.nature.com/articles/nature16526" }, { "title": "Enhanced proofreading governs CRISPR–Cas9 targeting accuracy", "url": "https://www.nature.com/articles/nature24268" }, { "title": "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage", "url": "https://pubmed.ncbi.nlm.nih.gov/27096365/" } ], "license": "https://creativecommons.org/licenses/by/4.0/" }