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Protein

CRISPR-associated endonuclease Cas9/Csn1

Gene

cas9

Organism
Streptococcus pyogenes serotype M1
Status
Reviewed-Annotation score: -Experimental evidence at protein leveli

Functioni

CRISPR (clustered regularly interspaced short palindromic repeat) is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids) (PubMed:21455174). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and this protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA; Cas9 only stabilizes the pre-crRNA:tracrRNA interaction and has no catalytic function in RNA processing (PubMed:24270795). Subsequently Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer; Cas9 is inactive in the absence of the 2 guide RNAs (gRNA). The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3'-5' exonucleolytically. DNA-binding requires protein and both gRNAs, as does nuclease activity. Cas9 recognizes the protospacer adjacent motif (PAM) in the CRISPR repeat sequences to help distinguish self versus nonself, as targets within the bacterial CRISPR locus do not have PAMs. DNA strand separation and heteroduplex formation starts at PAM sites; PAM recognition is required for catalytic activity (PubMed:24476820). Confers immunity against a plasmid with homology to the appropriate CRISPR spacer sequences (CRISPR interference) (PubMed:21455174).6 Publications

Cofactori

Mg2+2 PublicationsNote: Endonuclease activity on target dsDNA requires Mg2+ (PubMed:22745249). The RuvC-like nuclease domain should have 2 divalent cations, while the HNH domain should have 1. Crystals are often soaked in MgCl(2) or MnCl2+.3 Publications

Activity regulationi

Only has nuclease activity when bound to both gRNAs (crRNA plus tracrRNA), which results in conformational changes in the protein and formation of a central channel which binds target DNA (PubMed:24505130). Also requires interaction with PAM to trigger catalytic activity (PubMed:24476820). Nuclease activity is inhibited by EDTA (PubMed:26841432).4 Publications

Sites

Feature keyPosition(s)DescriptionActionsGraphical viewLength
Active sitei10For RuvC-like nuclease domain1 Publication1
Metal bindingi10Manganese 11 Publication1
Metal bindingi10Manganese 21 Publication1
Metal bindingi762Manganese 11 Publication1
Metal bindingi766Manganese 11 Publication1
Metal bindingi766Manganese 21 Publication1
Active sitei840Proton acceptor for HNH nuclease domain1 Publication1
Metal bindingi983Manganese 2; via pros nitrogen1 Publication1
Metal bindingi1297Manganese 3; via tele nitrogen1 Publication1
Metal bindingi1328Manganese 31 Publication1

GO - Molecular functioni

GO - Biological processi

Keywordsi

Molecular functionDNA-binding, Endonuclease, Exonuclease, Hydrolase, Nuclease, RNA-binding
Biological processAntiviral defense
LigandMagnesium, Manganese, Metal-binding

Names & Taxonomyi

Protein namesi
Recommended name:
CRISPR-associated endonuclease Cas9/Csn1UniRule annotation (EC:3.1.-.-UniRule annotation)
Alternative name(s):
SpCas91 Publication
SpyCas91 Publication
Gene namesi
Name:cas91 PublicationUniRule annotation
Synonyms:csn11 Publication
Ordered Locus Names:SPy_1046
OrganismiStreptococcus pyogenes serotype M1
Taxonomic identifieri301447 [NCBI]
Taxonomic lineageiBacteriaFirmicutesBacilliLactobacillalesStreptococcaceaeStreptococcus
Proteomesi
  • UP000000750 Componenti: Chromosome

Pathology & Biotechi

Biotechnological usei

Coexpression of Cas9 with an artifical single guide RNA (sgRNA) which fuses the crRNA with the tracrRNA in human cells has shown it is possible to target and modify DNA sequences of interest (PubMed:23287722, PubMed:23360966, PubMed:23386978). Cas9 plus the 2 sgRNAs have also been expressed individually in human and mouse cells to achieve DNA targeting; cleavage efficiencies of the artificial sgRNA were lower that those for systems with the 2 sgRNAs expressed separately (PubMed:23287718). Microinjection of Cas9-encoding mRNA and a synthetic sgRNA into zebrafish embryos induces targeted mutations (PubMed:23360964). In all cases introduction of multiple sgRNAs leads to multiplexed editing of genomic loci; DNA has also been inserted into a mammalian locus of interest. In S.pneumoniae and E.coli it has been used to generate markerless mutations; mutiple changes can be made simultaneously (PubMed:23360965). Studies to make mutations that alter the PAM-specificity and thus recognition possibilities have been made, but are not annotated in this database (PubMed:26098369).7 Publications

Disruption phenotypei

Loss of correct processing of pre-crRNA and tracrRNA. Loss of immunity against a plasmid with homology to CRISPR spacer sequences.1 Publication

Mutagenesis

Feature keyPosition(s)DescriptionActionsGraphical viewLength
Mutagenesisi10D → A: Target DNA noncomplementary to the crRNA is not cleaved; nickase activity. Processes guide RNAs. In vivo, loss of Cas9-mediated CRISPR interference in plasmid transformation. Able to bind guide RNAs and target DNA but not cleave DNA; when associated with A-840. 3 Publications1
Mutagenesisi15S → A: Decreased DNA cleavage. 1 Publication1
Mutagenesisi66R → A: Significantly decreased DNA cleavage. 1 Publication1
Mutagenesisi70R → A: No DNA cleavage. 1 Publication1
Mutagenesisi74R → A: Significantly decreased DNA cleavage. 1 Publication1
Mutagenesisi78R → A: Moderately decreased DNA cleavage. 1 Publication1
Mutagenesisi97 – 150Missing : No nuclease activity. 1 PublicationAdd BLAST54
Mutagenesisi165R → A: Moderately decreased DNA cleavage. 1 Publication1
Mutagenesisi175 – 307Missing : About 50% nuclease activity. 1 PublicationAdd BLAST133
Mutagenesisi312 – 409Missing : No nuclease activity. 1 PublicationAdd BLAST98
Mutagenesisi475 – 477PWN → AAA: Slight decrease in target DNA cleavage and DNA-binding. Almost complete loss of DNA cleavage and binding; when associated with 1125-A--A-1127. 1 Publication3
Mutagenesisi762E → A: Only cleaves 1 DNA strand, probably the noncomplementary strand. Processes guide RNAs correctly. In vivo, loss of Cas9-mediated CRISPR interference in plasmid transformation. 2 Publications1
Mutagenesisi840H → A: Target DNA complementary to the crRNA is not cleaved; nickase activity. In vivo, loss of Cas9-mediated CRISPR interference in plasmid transformation. Able to process and bind guide RNAs and target DNA but not cleave DNA; when associated with A-10. 4 Publications1
Mutagenesisi854N → A: Decreased DNA cleavage. Processes guide RNAs correctly. In vivo, retains Cas9-mediated CRISPR interference in plasmid transformation. 2 Publications1
Mutagenesisi863N → A: Only cleaves 1 DNA strand, probably the complementary strand. Processes guide RNAs correctly. In vivo, loss of Cas9-mediated CRISPR interference in plasmid transformation. 2 Publications1
Mutagenesisi982 – 983HH → AA: Processes guide RNAs correctly. 1 Publication2
Mutagenesisi982H → A: Decreased DNA cleavage. In vivo, loss of Cas9-mediated CRISPR interference in plasmid transformation. 1 Publication1
Mutagenesisi983H → A: Only cleaves 1 DNA strand, probably the noncomplementary strand. 1 Publication1
Mutagenesisi986D → A: Only cleaves 1 DNA strand, probably the noncomplementary strand. Processes guide RNAs correctly. In vivo, loss of Cas9-mediated CRISPR interference in plasmid transformation. 2 Publications1
Mutagenesisi1099 – 1368Missing : No nuclease activity. 1 PublicationAdd BLAST270
Mutagenesisi1125 – 1127DWD → AAA: No change in target DNA cleavage, slight decrease in DNA-binding. Almost complete loss of DNA cleavage and binding; when associated with 475-A--A-477. 1 Publication3
Mutagenesisi1132G → C: Probably inactivates protein. 1 Publication1
Mutagenesisi1333 – 1335RKR → AKA: Nearly complete loss of target DNA cleavage. 1 Publication3
Mutagenesisi1333R → A: Dramatically reduced target DNA binding, slightly decreased target cleavage. 1 Publication1
Mutagenesisi1335R → A: Dramatically reduced target DNA binding, slightly decreased target cleavage. 1 Publication1

PTM / Processingi

Molecule processing

Feature keyPosition(s)DescriptionActionsGraphical viewLength
ChainiPRO_00004184371 – 1368CRISPR-associated endonuclease Cas9/Csn1Add BLAST1368

Proteomic databases

PaxDbiQ99ZW2
PRIDEiQ99ZW2

Interactioni

Subunit structurei

Monomer. Binds crRNA and tracrRNA.UniRule annotation2 Publications

Protein-protein interaction databases

DIPiDIP-61504N
IntActiQ99ZW2, 3 interactors
STRINGi160490.SPy_1046

Structurei

Secondary structure

11368
Legend: HelixTurnBeta strandPDB Structure known for this area
Show more details

3D structure databases

ProteinModelPortaliQ99ZW2
SMRiQ99ZW2
ModBaseiSearch...
MobiDBiSearch...

Family & Domainsi

Domains and Repeats

Feature keyPosition(s)DescriptionActionsGraphical viewLength
Domaini770 – 921HNH Cas9-typePROSITE-ProRule annotationAdd BLAST152

Region

Feature keyPosition(s)DescriptionActionsGraphical viewLength
Regioni1 – 62RuvC-I1 PublicationAdd BLAST62
Regioni56 – 718Recognition lobe1 PublicationAdd BLAST663
Regioni56 – 73ARM1 PublicationAdd BLAST18
Regioni718 – 765RuvC-II1 PublicationAdd BLAST48
Regioni925 – 1102RuvC-III1 PublicationAdd BLAST178
Regioni1099 – 1368PAM-interacting domain (PI)1 PublicationAdd BLAST270

Motif

Feature keyPosition(s)DescriptionActionsGraphical viewLength
Motifi1333 – 1335PAM substrate-binding1 Publication3

Domaini

Has 2 endonuclease domains. The discontinuous RuvC-like domain (approximately residues 1-62, 718-765 and 925-1102) recognizes and cleaves the target DNA noncomplementary to crRNA while the HNH nuclease domain (residues 810-872) cleaves the target DNA complementary to crRNA (PubMed:22745249, PubMed:24529477).2 Publications
Has a bilobed architecture with a recognition lobe (REC, residues 60-718) and a discontinuous nuclease lobe (NUC, residues 1-59 and 719-1368) (PubMed:24529477, PubMed:24505130). The crRNA-target DNA lies in a channel between the 2 lobes (PubMed:24529477, PubMed:26841432). Binding of sgRNA induces large conformational changes further enhanced by target DNA binding (PubMed:26113724, PubMed:26841432). REC recognizes and binds differing regions of an artifical sgRNA in a sequence-independent manner. Deletions of parts of this lobe abolish nuclease activity (PubMed:24529477).4 Publications
The PAM-interacting domain (PI domain, approximately residues 1099-1368) recognizes the PAM motif; swapping the PI domain of this enzyme with that from S.thermophilus St3Cas9 (AC Q03JI6) prevents cleavage of DNA with the endogenous PAM site (5'-NGG-3') but confers the ability to cleave DNA with the PAM site specific for St3 CRISPRs.1 Publication

Sequence similaritiesi

Phylogenomic databases

eggNOGiCOG3513 LUCA
HOGENOMiHOG000071789
KOiK09952

Family and domain databases

HAMAPiMF_01480 Cas9, 1 hit
InterProiView protein in InterPro
IPR028629 Cas9
IPR032239 Cas9-BH
IPR032237 Cas9_PI
IPR032240 Cas9_REC
IPR033114 HNH_CAS9
IPR003615 HNH_nuc
PfamiView protein in Pfam
PF16593 Cas9-BH, 1 hit
PF16595 Cas9_PI, 1 hit
PF16592 Cas9_REC, 1 hit
PF13395 HNH_4, 1 hit
TIGRFAMsiTIGR01865 cas_Csn1, 1 hit
PROSITEiView protein in PROSITE
PS51749 HNH_CAS9, 1 hit

Sequencei

Sequence statusi: Complete.

Q99ZW2-1 [UniParc]FASTAAdd to basket
« Hide
        10         20         30         40         50
MDKKYSIGLD IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA
60 70 80 90 100
LLFDSGETAE ATRLKRTARR RYTRRKNRIC YLQEIFSNEM AKVDDSFFHR
110 120 130 140 150
LEESFLVEED KKHERHPIFG NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD
160 170 180 190 200
LRLIYLALAH MIKFRGHFLI EGDLNPDNSD VDKLFIQLVQ TYNQLFEENP
210 220 230 240 250
INASGVDAKA ILSARLSKSR RLENLIAQLP GEKKNGLFGN LIALSLGLTP
260 270 280 290 300
NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA QIGDQYADLF LAAKNLSDAI
310 320 330 340 350
LLSDILRVNT EITKAPLSAS MIKRYDEHHQ DLTLLKALVR QQLPEKYKEI
360 370 380 390 400
FFDQSKNGYA GYIDGGASQE EFYKFIKPIL EKMDGTEELL VKLNREDLLR
410 420 430 440 450
KQRTFDNGSI PHQIHLGELH AILRRQEDFY PFLKDNREKI EKILTFRIPY
460 470 480 490 500
YVGPLARGNS RFAWMTRKSE ETITPWNFEE VVDKGASAQS FIERMTNFDK
510 520 530 540 550
NLPNEKVLPK HSLLYEYFTV YNELTKVKYV TEGMRKPAFL SGEQKKAIVD
560 570 580 590 600
LLFKTNRKVT VKQLKEDYFK KIECFDSVEI SGVEDRFNAS LGTYHDLLKI
610 620 630 640 650
IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLKTYA HLFDDKVMKQ
660 670 680 690 700
LKRRRYTGWG RLSRKLINGI RDKQSGKTIL DFLKSDGFAN RNFMQLIHDD
710 720 730 740 750
SLTFKEDIQK AQVSGQGDSL HEHIANLAGS PAIKKGILQT VKVVDELVKV
760 770 780 790 800
MGRHKPENIV IEMARENQTT QKGQKNSRER MKRIEEGIKE LGSQILKEHP
810 820 830 840 850
VENTQLQNEK LYLYYLQNGR DMYVDQELDI NRLSDYDVDH IVPQSFLKDD
860 870 880 890 900
SIDNKVLTRS DKNRGKSDNV PSEEVVKKMK NYWRQLLNAK LITQRKFDNL
910 920 930 940 950
TKAERGGLSE LDKAGFIKRQ LVETRQITKH VAQILDSRMN TKYDENDKLI
960 970 980 990 1000
REVKVITLKS KLVSDFRKDF QFYKVREINN YHHAHDAYLN AVVGTALIKK
1010 1020 1030 1040 1050
YPKLESEFVY GDYKVYDVRK MIAKSEQEIG KATAKYFFYS NIMNFFKTEI
1060 1070 1080 1090 1100
TLANGEIRKR PLIETNGETG EIVWDKGRDF ATVRKVLSMP QVNIVKKTEV
1110 1120 1130 1140 1150
QTGGFSKESI LPKRNSDKLI ARKKDWDPKK YGGFDSPTVA YSVLVVAKVE
1160 1170 1180 1190 1200
KGKSKKLKSV KELLGITIME RSSFEKNPID FLEAKGYKEV KKDLIIKLPK
1210 1220 1230 1240 1250
YSLFELENGR KRMLASAGEL QKGNELALPS KYVNFLYLAS HYEKLKGSPE
1260 1270 1280 1290 1300
DNEQKQLFVE QHKHYLDEII EQISEFSKRV ILADANLDKV LSAYNKHRDK
1310 1320 1330 1340 1350
PIREQAENII HLFTLTNLGA PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ
1360
SITGLYETRI DLSQLGGD
Length:1,368
Mass (Da):158,441
Last modified:June 1, 2001 - v1
Checksum:i07D04F0B5965762F
GO

Sequence databases

Select the link destinations:
EMBLi
GenBanki
DDBJi
Links Updated
AE004092 Genomic DNA Translation: AAK33936.1
RefSeqiNP_269215.1, NC_002737.2

Genome annotation databases

EnsemblBacteriaiAAK33936; AAK33936; SPy_1046
GeneIDi901176
KEGGispy:SPy_1046
PATRICifig|160490.10.peg.902

Similar proteinsi

Cross-referencesi

Sequence databases

Select the link destinations:
EMBLi
GenBanki
DDBJi
Links Updated
AE004092 Genomic DNA Translation: AAK33936.1
RefSeqiNP_269215.1, NC_002737.2

3D structure databases

Select the link destinations:
PDBei
RCSB PDBi
PDBji
Links Updated
PDB entryMethodResolution (Å)ChainPositionsPDBsum
4CMPX-ray2.62A/B1-1368[»]
4CMQX-ray3.09A/B1-1368[»]
4OO8X-ray2.50A/D1-1368[»]
4UN3X-ray2.59B1-1368[»]
4UN4X-ray2.37B1-1368[»]
4UN5X-ray2.40B1-1368[»]
4ZT0X-ray2.90A/C1-1368[»]
4ZT9X-ray3.10A/C1-1368[»]
5B2RX-ray2.00B1-1368[»]
5B2SX-ray2.20B1-1368[»]
5B2TX-ray2.20B1-1368[»]
5F9RX-ray3.40B1-1368[»]
5FQ5X-ray2.14B1-1368[»]
5FW1X-ray2.50B1-1368[»]
5FW2X-ray2.68B1-1368[»]
5FW3X-ray2.70B1-1368[»]
5VW1X-ray2.60A1-1368[»]
5VZLelectron microscopy3.90A1-1368[»]
5XBLX-ray3.05A1-1368[»]
5Y36electron microscopy5.20A1-1368[»]
ProteinModelPortaliQ99ZW2
SMRiQ99ZW2
ModBaseiSearch...
MobiDBiSearch...

Protein-protein interaction databases

DIPiDIP-61504N
IntActiQ99ZW2, 3 interactors
STRINGi160490.SPy_1046

Proteomic databases

PaxDbiQ99ZW2
PRIDEiQ99ZW2

Protocols and materials databases

Structural Biology KnowledgebaseSearch...

Genome annotation databases

EnsemblBacteriaiAAK33936; AAK33936; SPy_1046
GeneIDi901176
KEGGispy:SPy_1046
PATRICifig|160490.10.peg.902

Phylogenomic databases

eggNOGiCOG3513 LUCA
HOGENOMiHOG000071789
KOiK09952

Family and domain databases

HAMAPiMF_01480 Cas9, 1 hit
InterProiView protein in InterPro
IPR028629 Cas9
IPR032239 Cas9-BH
IPR032237 Cas9_PI
IPR032240 Cas9_REC
IPR033114 HNH_CAS9
IPR003615 HNH_nuc
PfamiView protein in Pfam
PF16593 Cas9-BH, 1 hit
PF16595 Cas9_PI, 1 hit
PF16592 Cas9_REC, 1 hit
PF13395 HNH_4, 1 hit
TIGRFAMsiTIGR01865 cas_Csn1, 1 hit
PROSITEiView protein in PROSITE
PS51749 HNH_CAS9, 1 hit
ProtoNetiSearch...

Entry informationi

Entry nameiCAS9_STRP1
AccessioniPrimary (citable) accession number: Q99ZW2
Entry historyiIntegrated into UniProtKB/Swiss-Prot: July 11, 2012
Last sequence update: June 1, 2001
Last modified: November 7, 2018
This is version 104 of the entry and version 1 of the sequence. See complete history.
Entry statusiReviewed (UniProtKB/Swiss-Prot)
Annotation programProkaryotic Protein Annotation Program

Miscellaneousi

Keywords - Technical termi

3D-structure, Complete proteome

Documents

  1. SIMILARITY comments
    Index of protein domains and families
  2. PDB cross-references
    Index of Protein Data Bank (PDB) cross-references
UniProt is an ELIXIR core data resource
Main funding by: National Institutes of Health

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