CAS9_STAAU
ID CAS9_STAAU Reviewed; 1053 AA.
AC J7RUA5;
DT 11-MAY-2016, integrated into UniProtKB/Swiss-Prot.
DT 31-OCT-2012, sequence version 1.
DT 03-AUG-2022, entry version 42.
DE RecName: Full=CRISPR-associated endonuclease Cas9 {ECO:0000255|HAMAP-Rule:MF_01480};
DE EC=3.1.-.- {ECO:0000255|HAMAP-Rule:MF_01480};
DE AltName: Full=SaCas9 {ECO:0000303|PubMed:25830891};
GN Name=cas9 {ECO:0000255|HAMAP-Rule:MF_01480};
OS Staphylococcus aureus.
OC Bacteria; Firmicutes; Bacilli; Bacillales; Staphylococcaceae;
OC Staphylococcus.
OX NCBI_TaxID=1280;
RN [1]
RP NUCLEOTIDE SEQUENCE [GENOMIC DNA].
RC STRAIN=M06/0171;
RX PubMed=23147725; DOI=10.1128/aac.01689-12;
RA Kinnevey P.M., Shore A.C., Brennan G.I., Sullivan D.J., Ehricht R.,
RA Monecke S., Slickers P., Coleman D.C.;
RT "Emergence of sequence type 779 methicillin-resistant Staphylococcus aureus
RT harboring a novel pseudo Staphylococcal cassette chromosome mec (SCCmec)-
RT SCC-SCCCRISPR composite element in Irish hospitals.";
RL Antimicrob. Agents Chemother. 57:524-531(2013).
RN [2]
RP FUNCTION, BIOTECHNOLOGY IN HUMAN CELLS AND MICE, AND DNA-BINDING.
RX PubMed=25830891; DOI=10.1038/nature14299;
RA Ran F.A., Cong L., Yan W.X., Scott D.A., Gootenberg J.S., Kriz A.J.,
RA Zetsche B., Shalem O., Wu X., Makarova K.S., Koonin E.V., Sharp P.A.,
RA Zhang F.;
RT "In vivo genome editing using Staphylococcus aureus Cas9.";
RL Nature 520:186-191(2015).
RN [3]
RP FUNCTION, AND BIOTECHNOLOGY IN HUMAN CELLS.
RX PubMed=26098369; DOI=10.1038/nature14592;
RA Kleinstiver B.P., Prew M.S., Tsai S.Q., Topkar V.V., Nguyen N.T., Zheng Z.,
RA Gonzales A.P., Li Z., Peterson R.T., Yeh J.R., Aryee M.J., Joung J.K.;
RT "Engineered CRISPR-Cas9 nucleases with altered PAM specificities.";
RL Nature 523:481-485(2015).
RN [4]
RP X-RAY CRYSTALLOGRAPHY (2.60 ANGSTROMS) IN COMPLEX WITH SGRNA AND
RP SINGLE-STRAND TARGET DNA, ACTIVE SITES, DOMAIN, BIOTECHNOLOGY, MUTAGENESIS
RP OF ASP-10; GLU-477; HIS-557; ASN-580; HIS-701; ASP-704; THR-787; ASN-985;
RP ASN-986; ARG-991; GLU-993 AND ARG-1015, DNA-BINDING, AND RNA-BINDING.
RX PubMed=26317473; DOI=10.1016/j.cell.2015.08.007;
RA Nishimasu H., Cong L., Yan W.X., Ran F.A., Zetsche B., Li Y.,
RA Kurabayashi A., Ishitani R., Zhang F., Nureki O.;
RT "Crystal structure of Staphylococcus aureus Cas9.";
RL Cell 162:1113-1126(2015).
CC -!- FUNCTION: CRISPR (clustered regularly interspaced short palindromic
CC repeat) is an adaptive immune system that provides protection against
CC mobile genetic elements (viruses, transposable elements and conjugative
CC plasmids). CRISPR clusters contain spacers, sequences complementary to
CC antecedent mobile elements, and target invading nucleic acids. CRISPR
CC clusters are transcribed and processed into CRISPR RNA (crRNA). In type
CC II CRISPR systems correct processing of pre-crRNA requires a trans-
CC encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and this
CC protein. The tracrRNA serves as a guide for ribonuclease 3-aided
CC processing of pre-crRNA. Subsequently Cas9/crRNA/tracrRNA
CC endonucleolytically cleaves linear or circular dsDNA target
CC complementary to the spacer; Cas9 is inactive in the absence of the 2
CC guide RNAs (gRNA). Cas9 recognizes the protospacer adjacent motif (PAM)
CC in the CRISPR repeat sequences to help distinguish self versus nonself,
CC as targets within the bacterial CRISPR locus do not have PAMs. PAM
CC recognition is also required for catalytic activity.
CC {ECO:0000255|HAMAP-Rule:MF_01480, ECO:0000269|PubMed:25830891,
CC ECO:0000269|PubMed:26098369}.
CC -!- COFACTOR:
CC Name=Mg(2+); Xref=ChEBI:CHEBI:18420;
CC Evidence={ECO:0000255|HAMAP-Rule:MF_01480};
CC -!- SUBUNIT: Monomer. Binds crRNA and tracrRNA. {ECO:0000255|HAMAP-
CC Rule:MF_01480}.
CC -!- DOMAIN: Has a bilobed architecture with a recognition lobe (REC,
CC residues 41-425) and a discontinuous nuclease lobe (NUC, residues 1-40
CC and 453-1053); the crRNA-target DNA lies in a channel between the 2
CC lobes (PubMed:26317473). The NUC lobe has 2 endonuclease domains. The
CC discontinuous RuvC-like domain in NUC cleaves the target DNA
CC noncomplementary to crRNA while the HNH nuclease domain cleaves the
CC target DNA complementary to crRNA (PubMed:26317473).
CC {ECO:0000255|HAMAP-Rule:MF_01480, ECO:0000269|PubMed:26317473}.
CC -!- BIOTECHNOLOGY: Coexpression of Cas9 with an artificial sgRNA which
CC fuses crRNA with the tracrRNA in human cells has shown it is possible
CC to target and modify DNA sequences of interest (PubMed:25830891,
CC PubMed:26098369). The smaller size of Cas9 from this organism (1053
CC residues versus 1368 for Streptococcus pyogenes) makes it easier to
CC package the gene in an adeno-associated virus for delivery to 5-6 week
CC old C57/BL6 mice; 2 different target genes were modifed as desired
CC (PubMed:25830891). {ECO:0000269|PubMed:25830891,
CC ECO:0000269|PubMed:26098369}.
CC -!- SIMILARITY: Belongs to the CRISPR-associated Cas9 family. Subtype II-A
CC subfamily. {ECO:0000255|HAMAP-Rule:MF_01480}.
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DR EMBL; HE980450; CCK74173.1; -; Genomic_DNA.
DR PDB; 5AXW; X-ray; 2.70 A; A=1-1053.
DR PDB; 5CZZ; X-ray; 2.60 A; A=1-1053.
DR PDB; 7EL1; X-ray; 2.22 A; A=1-1053.
DR PDBsum; 5AXW; -.
DR PDBsum; 5CZZ; -.
DR PDBsum; 7EL1; -.
DR AlphaFoldDB; J7RUA5; -.
DR SMR; J7RUA5; -.
DR PRIDE; J7RUA5; -.
DR GO; GO:0003677; F:DNA binding; IEA:UniProtKB-KW.
DR GO; GO:0004519; F:endonuclease activity; IEA:UniProtKB-UniRule.
DR GO; GO:0046872; F:metal ion binding; IEA:UniProtKB-UniRule.
DR GO; GO:0003723; F:RNA binding; IEA:UniProtKB-KW.
DR GO; GO:0051607; P:defense response to virus; IEA:UniProtKB-UniRule.
DR GO; GO:0043571; P:maintenance of CRISPR repeat elements; IEA:UniProtKB-UniRule.
DR Gene3D; 3.30.420.10; -; 3.
DR HAMAP; MF_01480; Cas9; 1.
DR InterPro; IPR028629; Cas9.
DR InterPro; IPR040555; Cas9_PI2.
DR InterPro; IPR040656; Cas9_WED_dom.
DR InterPro; IPR033114; HNH_CAS9.
DR InterPro; IPR003615; HNH_nuc.
DR InterPro; IPR036397; RNaseH_sf.
DR InterPro; IPR041383; RuvC_III.
DR Pfam; PF18070; Cas9_PI2; 1.
DR Pfam; PF18061; CRISPR_Cas9_WED; 1.
DR Pfam; PF13395; HNH_4; 1.
DR Pfam; PF18541; RuvC_III; 1.
DR TIGRFAMs; TIGR01865; cas_Csn1; 1.
DR PROSITE; PS51749; HNH_CAS9; 1.
PE 1: Evidence at protein level;
KW 3D-structure; Antiviral defense; DNA-binding; Endonuclease; Hydrolase;
KW Magnesium; Manganese; Metal-binding; Nuclease; RNA-binding.
FT CHAIN 1..1053
FT /note="CRISPR-associated endonuclease Cas9"
FT /id="PRO_0000436103"
FT DOMAIN 480..646
FT /note="HNH Cas9-type"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_01480"
FT REGION 1..41
FT /note="RuvC-I"
FT /evidence="ECO:0000305|PubMed:26317473"
FT REGION 41..426
FT /note="Recognition lobe"
FT /evidence="ECO:0000305|PubMed:26317473"
FT REGION 435..481
FT /note="RuvC-II"
FT /evidence="ECO:0000305|PubMed:26317473"
FT REGION 650..775
FT /note="RuvC-III"
FT /evidence="ECO:0000305|PubMed:26317473"
FT REGION 882..889
FT /note="PAM substrate-binding"
FT /evidence="ECO:0000269|PubMed:26317473"
FT REGION 910..1053
FT /note="PAM-interacting domain (PI)"
FT /evidence="ECO:0000305|PubMed:26317473"
FT REGION 985..993
FT /note="PAM substrate-binding"
FT /evidence="ECO:0000269|PubMed:26317473"
FT ACT_SITE 10
FT /note="For RuvC-like nuclease domain"
FT /evidence="ECO:0000305|PubMed:26317473"
FT ACT_SITE 557
FT /note="Proton acceptor for HNH nuclease domain"
FT /evidence="ECO:0000305|PubMed:26317473"
FT BINDING 10
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /ligand_label="1"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_01480"
FT BINDING 10
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /ligand_label="2"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_01480"
FT BINDING 477
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /ligand_label="1"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_01480"
FT BINDING 481
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /ligand_label="1"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_01480"
FT BINDING 481
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /ligand_label="2"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_01480"
FT BINDING 701
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /ligand_label="2"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_01480"
FT BINDING 789
FT /ligand="RNA"
FT /ligand_id="ChEBI:CHEBI:33697"
FT /ligand_part="PAM RNA"
FT /evidence="ECO:0000269|PubMed:26317473"
FT MUTAGEN 10
FT /note="D->A: Target DNA not cleaved."
FT /evidence="ECO:0000269|PubMed:26317473"
FT MUTAGEN 477
FT /note="E->A: Target DNA not cleaved."
FT /evidence="ECO:0000269|PubMed:26317473"
FT MUTAGEN 557
FT /note="H->A: Target DNA not cleaved."
FT /evidence="ECO:0000269|PubMed:26317473"
FT MUTAGEN 580
FT /note="N->A: Target DNA not cleaved."
FT /evidence="ECO:0000269|PubMed:26317473"
FT MUTAGEN 701
FT /note="H->A: Target DNA not cleaved."
FT /evidence="ECO:0000269|PubMed:26317473"
FT MUTAGEN 704
FT /note="D->A: Target DNA not cleaved."
FT /evidence="ECO:0000269|PubMed:26317473"
FT MUTAGEN 787
FT /note="T->A: 60% target DNA cleaved."
FT /evidence="ECO:0000269|PubMed:26317473"
FT MUTAGEN 985
FT /note="N->A: 40% target DNA cleaved."
FT /evidence="ECO:0000269|PubMed:26317473"
FT MUTAGEN 986
FT /note="N->A: 75% target DNA cleaved."
FT /evidence="ECO:0000269|PubMed:26317473"
FT MUTAGEN 991
FT /note="R->A: 20% target DNA cleaved."
FT /evidence="ECO:0000269|PubMed:26317473"
FT MUTAGEN 993
FT /note="E->A: 50% target DNA cleaved."
FT /evidence="ECO:0000269|PubMed:26317473"
FT MUTAGEN 1015
FT /note="R->A: 5% target DNA cleaved."
FT /evidence="ECO:0000269|PubMed:26317473"
FT STRAND 5..11
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 13..22
FT /evidence="ECO:0007829|PDB:7EL1"
FT TURN 23..25
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 28..35
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 41..73
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 88..95
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 102..114
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 118..121
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 130..132
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 134..144
FT /evidence="ECO:0007829|PDB:7EL1"
FT TURN 145..147
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 150..161
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 167..169
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 173..187
FT /evidence="ECO:0007829|PDB:7EL1"
FT TURN 188..190
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 195..206
FT /evidence="ECO:0007829|PDB:7EL1"
FT TURN 211..213
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 226..233
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 251..263
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 269..271
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 277..286
FT /evidence="ECO:0007829|PDB:7EL1"
FT TURN 287..290
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 296..303
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 307..309
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 317..319
FT /evidence="ECO:0007829|PDB:5CZZ"
FT HELIX 327..335
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 339..342
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 345..357
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 361..369
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 377..383
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 397..409
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 414..420
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 438..440
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 441..444
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 448..468
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 472..478
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 486..493
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 499..511
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 515..517
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 518..529
FT /evidence="ECO:0007829|PDB:7EL1"
FT TURN 530..532
FT /evidence="ECO:0007829|PDB:7EL1"
FT TURN 535..537
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 543..548
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 550..552
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 553..559
FT /evidence="ECO:0007829|PDB:7EL1"
FT TURN 561..563
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 571..576
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 577..583
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 588..592
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 594..596
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 601..611
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 612..614
FT /evidence="ECO:0007829|PDB:5CZZ"
FT HELIX 620..626
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 635..644
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 651..666
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 672..675
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 680..686
FT /evidence="ECO:0007829|PDB:7EL1"
FT TURN 687..689
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 699..722
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 744..755
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 758..764
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 769..773
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 779..781
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 789..793
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 799..805
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 815..822
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 824..826
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 828..831
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 834..846
FT /evidence="ECO:0007829|PDB:7EL1"
FT TURN 847..849
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 853..861
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 880..886
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 894..896
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 904..906
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 911..919
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 922..929
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 930..932
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 933..935
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 937..942
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 944..953
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 961..966
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 971..974
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 977..986
FT /evidence="ECO:0007829|PDB:7EL1"
FT TURN 987..990
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 991..995
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 997..999
FT /evidence="ECO:0007829|PDB:7EL1"
FT HELIX 1001..1007
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 1016..1019
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 1027..1032
FT /evidence="ECO:0007829|PDB:7EL1"
FT STRAND 1038..1040
FT /evidence="ECO:0007829|PDB:7EL1"
SQ SEQUENCE 1053 AA; 123949 MW; E38BF9FF0AAC0A60 CRC64;
MKRNYILGLD IGITSVGYGI IDYETRDVID AGVRLFKEAN VENNEGRRSK RGARRLKRRR
RHRIQRVKKL LFDYNLLTDH SELSGINPYE ARVKGLSQKL SEEEFSAALL HLAKRRGVHN
VNEVEEDTGN ELSTKEQISR NSKALEEKYV AELQLERLKK DGEVRGSINR FKTSDYVKEA
KQLLKVQKAY HQLDQSFIDT YIDLLETRRT YYEGPGEGSP FGWKDIKEWY EMLMGHCTYF
PEELRSVKYA YNADLYNALN DLNNLVITRD ENEKLEYYEK FQIIENVFKQ KKKPTLKQIA
KEILVNEEDI KGYRVTSTGK PEFTNLKVYH DIKDITARKE IIENAELLDQ IAKILTIYQS
SEDIQEELTN LNSELTQEEI EQISNLKGYT GTHNLSLKAI NLILDELWHT NDNQIAIFNR
LKLVPKKVDL SQQKEIPTTL VDDFILSPVV KRSFIQSIKV INAIIKKYGL PNDIIIELAR
EKNSKDAQKM INEMQKRNRQ TNERIEEIIR TTGKENAKYL IEKIKLHDMQ EGKCLYSLEA
IPLEDLLNNP FNYEVDHIIP RSVSFDNSFN NKVLVKQEEN SKKGNRTPFQ YLSSSDSKIS
YETFKKHILN LAKGKGRISK TKKEYLLEER DINRFSVQKD FINRNLVDTR YATRGLMNLL
RSYFRVNNLD VKVKSINGGF TSFLRRKWKF KKERNKGYKH HAEDALIIAN ADFIFKEWKK
LDKAKKVMEN QMFEEKQAES MPEIETEQEY KEIFITPHQI KHIKDFKDYK YSHRVDKKPN
RELINDTLYS TRKDDKGNTL IVNNLNGLYD KDNDKLKKLI NKSPEKLLMY HHDPQTYQKL
KLIMEQYGDE KNPLYKYYEE TGNYLTKYSK KDNGPVIKKI KYYGNKLNAH LDITDDYPNS
RNKVVKLSLK PYRFDVYLDN GVYKFVTVKN LDVIKKENYY EVNSKCYEEA KKLKKISNQA
EFIASFYNND LIKINGELYR VIGVNNDLLN RIEVNMIDIT YREYLENMND KRPPRIIKTI
ASKTQSIKKY STDILGNLYE VKSKKHPQII KKG
