CAS9_STRTR
ID CAS9_STRTR Reviewed; 1409 AA.
AC G3ECR1;
DT 13-JUN-2012, integrated into UniProtKB/Swiss-Prot.
DT 13-JUN-2012, sequence version 2.
DT 03-AUG-2022, entry version 39.
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=St-Cas9;
GN Name=cas9 {ECO:0000255|HAMAP-Rule:MF_01480}; Synonyms=csn1;
OS Streptococcus thermophilus.
OC Bacteria; Firmicutes; Bacilli; Lactobacillales; Streptococcaceae;
OC Streptococcus.
OX NCBI_TaxID=1308;
RN [1]
RP NUCLEOTIDE SEQUENCE [GENOMIC DNA], FUNCTION IN PLASMID RESISTANCE,
RP EXPRESSION OF CRISPR3/CAS IN E.COLI, DISRUPTION PHENOTYPE, AND MUTAGENESIS
RP OF ASP-31; HIS-868; ASN-882 AND ASN-891.
RC STRAIN=DGCC7710;
RX PubMed=21813460; DOI=10.1093/nar/gkr606;
RA Sapranauskas R., Gasiunas G., Fremaux C., Barrangou R., Horvath P.,
RA Siksnys V.;
RT "The Streptococcus thermophilus CRISPR/Cas system provides immunity in
RT Escherichia coli.";
RL Nucleic Acids Res. 39:9275-9282(2011).
RN [2]
RP FUNCTION AS A DNA ENDONUCLEASE, COFACTOR, SUBUNIT, POSSIBLE BIOTECHNOLOGY,
RP DNA-BINDING, RNA-BINDING, EXPRESSION IN E.COLI, AND MUTAGENESIS OF ASP-31
RP AND ASN-891.
RC STRAIN=DGCC7710;
RX PubMed=22949671; DOI=10.1073/pnas.1208507109;
RA Gasiunas G., Barrangou R., Horvath P., Siksnys V.;
RT "Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for
RT adaptive immunity in bacteria.";
RL Proc. Natl. Acad. Sci. U.S.A. 109:E2579-E2586(2012).
RN [3]
RP FUNCTION, ACTIVITY REGULATION, TRACRRNA-BINDING, RNA-BINDING, AND
RP EXPRESSION IN E.COLI.
RC STRAIN=DGCC7710;
RX PubMed=23535272; DOI=10.4161/rna.24203;
RA Karvelis T., Gasiunas G., Miksys A., Barrangou R., Horvath P., Siksnys V.;
RT "crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus
RT thermophilus.";
RL RNA Biol. 10:841-851(2013).
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 Cas9.
CC The tracrRNA serves as a guide for ribonuclease 3-aided processing of
CC pre-crRNA (Probable). Cas9/crRNA/tracrRNA endonucleolytically cleaves
CC linear or circular dsDNA target complementary to the spacer yielding
CC blunt ends; Cas9 is inactive in the absence of the 2 guide RNAs (gRNA).
CC Cas9 recognizes a 3'-G-rich protospacer adjacent motif (PAM, TGGTG in
CC this organism) in the CRISPR repeat sequences to help distinguish self
CC versus nonself, as targets within the bacterial CRISPR locus do not
CC have PAMs. PAM recognition is also required for catalytic activity.
CC When the CRISPR3/cas system consisting of cas9-cas1-cas2-csn2-CRISPR3
CC or just cas9-CRISPR3 is expressed in E.coli it prevents plasmids
CC homologous to spacers 1 or 2 from transforming.
CC {ECO:0000269|PubMed:21813460, ECO:0000269|PubMed:22949671,
CC ECO:0000269|PubMed:23535272, ECO:0000305}.
CC -!- COFACTOR:
CC Name=Mg(2+); Xref=ChEBI:CHEBI:18420;
CC Evidence={ECO:0000269|PubMed:22949671};
CC Note=Endonuclease activity on target DNA requires Mg(2+).
CC {ECO:0000269|PubMed:22949671};
CC -!- ACTIVITY REGULATION: Only has nuclease activity when bound to both
CC gRNAs (crRNA plus tracrRNA). {ECO:0000269|PubMed:23535272}.
CC -!- SUBUNIT: Monomer (Probable). Binds crRNA and tracrRNA.
CC {ECO:0000255|HAMAP-Rule:MF_01480, ECO:0000269|PubMed:22949671,
CC ECO:0000305}.
CC -!- DOMAIN: Has 2 endonuclease domains. The discontinuous RuvC-like domain
CC cleaves the target DNA noncomplementary to crRNA while the HNH nuclease
CC domain cleaves the target DNA complementary to crRNA.
CC {ECO:0000255|HAMAP-Rule:MF_01480}.
CC -!- DISRUPTION PHENOTYPE: Plasmid transformation is restored.
CC {ECO:0000269|PubMed:21813460}.
CC -!- BIOTECHNOLOGY: The simplicity of the Cas9-gRNAs RNA-directed DNA
CC endonuclease activity may be used to target and modify a DNA sequence
CC of interest.
CC -!- MISCELLANEOUS: This strain encodes 4 CRISPR-Cas systems; this is
CC CRISPR3.
CC -!- SIMILARITY: Belongs to the CRISPR-associated protein Cas9 family.
CC Subtype II-A subfamily. {ECO:0000305}.
CC -!- CAUTION: It is uncertain whether Met-1 or Met-22 is the initiator.
CC {ECO:0000305}.
CC -!- SEQUENCE CAUTION:
CC Sequence=AEM62887.1; Type=Erroneous initiation; Note=Truncated N-terminus.; Evidence={ECO:0000305};
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DR EMBL; HQ712120; AEM62887.1; ALT_INIT; Genomic_DNA.
DR RefSeq; WP_024703962.1; NZ_WMLD01000001.1.
DR AlphaFoldDB; G3ECR1; -.
DR SMR; G3ECR1; -.
DR STRING; 322159.STER_1477; -.
DR PRIDE; G3ECR1; -.
DR eggNOG; COG3513; Bacteria.
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; -; 1.
DR HAMAP; MF_01480; Cas9; 1.
DR InterPro; IPR028629; Cas9.
DR InterPro; IPR032239; Cas9-BH.
DR InterPro; IPR032237; Cas9_PI.
DR InterPro; IPR032240; Cas9_REC.
DR InterPro; IPR033114; HNH_CAS9.
DR InterPro; IPR003615; HNH_nuc.
DR InterPro; IPR036397; RNaseH_sf.
DR Pfam; PF16593; Cas9-BH; 1.
DR Pfam; PF16595; Cas9_PI; 1.
DR Pfam; PF16592; Cas9_REC; 1.
DR Pfam; PF13395; HNH_4; 1.
DR TIGRFAMs; TIGR01865; cas_Csn1; 1.
DR PROSITE; PS51749; HNH_CAS9; 1.
PE 1: Evidence at protein level;
KW Antiviral defense; DNA-binding; Endonuclease; Hydrolase; Magnesium;
KW Manganese; Metal-binding; Nuclease; RNA-binding.
FT CHAIN 1..1409
FT /note="CRISPR-associated endonuclease Cas9"
FT /id="PRO_0000417879"
FT DOMAIN 792..949
FT /note="HNH Cas9-type"
FT /evidence="ECO:0000255|PROSITE-ProRule:PRU01085"
FT REGION 1121..1151
FT /note="Disordered"
FT /evidence="ECO:0000256|SAM:MobiDB-lite"
FT ACT_SITE 31
FT /note="For RuvC-like nuclease domain"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_01480"
FT ACT_SITE 868
FT /note="Proton acceptor for HNH nuclease domain"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_01480"
FT BINDING 31
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /ligand_label="1"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_01480"
FT BINDING 31
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /ligand_label="2"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_01480"
FT BINDING 784
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /ligand_label="1"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_01480"
FT BINDING 788
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /ligand_label="1"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_01480"
FT BINDING 788
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /ligand_label="2"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_01480"
FT BINDING 1011
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /ligand_label="2"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_01480"
FT MUTAGEN 31
FT /note="D->A: No longer prevents plasmid transformation.
FT Target DNA noncomplementary to the crRNA is not cleaved."
FT /evidence="ECO:0000269|PubMed:21813460,
FT ECO:0000269|PubMed:22949671"
FT MUTAGEN 868
FT /note="H->A: No longer prevents plasmid transformation.
FT Target DNA complementary to the crRNA is not cleaved."
FT /evidence="ECO:0000269|PubMed:21813460"
FT MUTAGEN 882
FT /note="N->A: No longer prevents plasmid transformation."
FT /evidence="ECO:0000269|PubMed:21813460"
FT MUTAGEN 891
FT /note="N->A: No longer prevents plasmid transformation."
FT /evidence="ECO:0000269|PubMed:21813460,
FT ECO:0000269|PubMed:22949671"
SQ SEQUENCE 1409 AA; 163571 MW; 6A511E727C853AA6 CRC64;
MLFNKCIIIS INLDFSNKEK CMTKPYSIGL DIGTNSVGWA VITDNYKVPS KKMKVLGNTS
KKYIKKNLLG VLLFDSGITA EGRRLKRTAR RRYTRRRNRI LYLQEIFSTE MATLDDAFFQ
RLDDSFLVPD DKRDSKYPIF GNLVEEKVYH DEFPTIYHLR KYLADSTKKA DLRLVYLALA
HMIKYRGHFL IEGEFNSKNN DIQKNFQDFL DTYNAIFESD LSLENSKQLE EIVKDKISKL
EKKDRILKLF PGEKNSGIFS EFLKLIVGNQ ADFRKCFNLD EKASLHFSKE SYDEDLETLL
GYIGDDYSDV FLKAKKLYDA ILLSGFLTVT DNETEAPLSS AMIKRYNEHK EDLALLKEYI
RNISLKTYNE VFKDDTKNGY AGYIDGKTNQ EDFYVYLKNL LAEFEGADYF LEKIDREDFL
RKQRTFDNGS IPYQIHLQEM RAILDKQAKF YPFLAKNKER IEKILTFRIP YYVGPLARGN
SDFAWSIRKR NEKITPWNFE DVIDKESSAE AFINRMTSFD LYLPEEKVLP KHSLLYETFN
VYNELTKVRF IAESMRDYQF LDSKQKKDIV RLYFKDKRKV TDKDIIEYLH AIYGYDGIEL
KGIEKQFNSS LSTYHDLLNI INDKEFLDDS SNEAIIEEII HTLTIFEDRE MIKQRLSKFE
NIFDKSVLKK LSRRHYTGWG KLSAKLINGI RDEKSGNTIL DYLIDDGISN RNFMQLIHDD
ALSFKKKIQK AQIIGDEDKG NIKEVVKSLP GSPAIKKGIL QSIKIVDELV KVMGGRKPES
IVVEMARENQ YTNQGKSNSQ QRLKRLEKSL KELGSKILKE NIPAKLSKID NNALQNDRLY
LYYLQNGKDM YTGDDLDIDR LSNYDIDHII PQAFLKDNSI DNKVLVSSAS NRGKSDDFPS
LEVVKKRKTF WYQLLKSKLI SQRKFDNLTK AERGGLLPED KAGFIQRQLV ETRQITKHVA
RLLDEKFNNK KDENNRAVRT VKIITLKSTL VSQFRKDFEL YKVREINDFH HAHDAYLNAV
IASALLKKYP KLEPEFVYGD YPKYNSFRER KSATEKVYFY SNIMNIFKKS ISLADGRVIE
RPLIEVNEET GESVWNKESD LATVRRVLSY PQVNVVKKVE EQNHGLDRGK PKGLFNANLS
SKPKPNSNEN LVGAKEYLDP KKYGGYAGIS NSFAVLVKGT IEKGAKKKIT NVLEFQGISI
LDRINYRKDK LNFLLEKGYK DIELIIELPK YSLFELSDGS RRMLASILST NNKRGEIHKG
NQIFLSQKFV KLLYHAKRIS NTINENHRKY VENHKKEFEE LFYYILEFNE NYVGAKKNGK
LLNSAFQSWQ NHSIDELCSS FIGPTGSERK GLFELTSRGS AADFEFLGVK IPRYRDYTPS
SLLKDATLIH QSVTGLYETR IDLAKLGEG
