CAS9B_STRTD
ID CAS9B_STRTD Reviewed; 1388 AA.
AC Q03JI6;
DT 06-MAR-2013, integrated into UniProtKB/Swiss-Prot.
DT 14-NOV-2006, sequence version 1.
DT 03-AUG-2022, entry version 78.
DE RecName: Full=CRISPR-associated endonuclease Cas9 2 {ECO:0000255|HAMAP-Rule:MF_01480};
DE EC=3.1.-.- {ECO:0000255|HAMAP-Rule:MF_01480};
DE AltName: Full=Cas9*;
DE AltName: Full=St3Cas9;
GN Name=cas9-2 {ECO:0000255|HAMAP-Rule:MF_01480}; Synonyms=csn1;
GN OrderedLocusNames=STER_1477;
OS Streptococcus thermophilus (strain ATCC BAA-491 / LMD-9).
OC Bacteria; Firmicutes; Bacilli; Lactobacillales; Streptococcaceae;
OC Streptococcus.
OX NCBI_TaxID=322159;
RN [1]
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=ATCC BAA-491 / LMD-9;
RX PubMed=17030793; DOI=10.1073/pnas.0607117103;
RA Makarova K.S., Slesarev A., Wolf Y.I., Sorokin A., Mirkin B., Koonin E.V.,
RA Pavlov A., Pavlova N., Karamychev V., Polouchine N., Shakhova V.,
RA Grigoriev I., Lou Y., Rohksar D., Lucas S., Huang K., Goodstein D.M.,
RA Hawkins T., Plengvidhya V., Welker D., Hughes J., Goh Y., Benson A.,
RA Baldwin K., Lee J.-H., Diaz-Muniz I., Dosti B., Smeianov V., Wechter W.,
RA Barabote R., Lorca G., Altermann E., Barrangou R., Ganesan B., Xie Y.,
RA Rawsthorne H., Tamir D., Parker C., Breidt F., Broadbent J.R., Hutkins R.,
RA O'Sullivan D., Steele J., Unlu G., Saier M.H. Jr., Klaenhammer T.,
RA Richardson P., Kozyavkin S., Weimer B.C., Mills D.A.;
RT "Comparative genomics of the lactic acid bacteria.";
RL Proc. Natl. Acad. Sci. U.S.A. 103:15611-15616(2006).
RN [2]
RP FUNCTION AS AN DNA ENDONUCLEASE, SUBUNIT, AND RNA-BINDING.
RC STRAIN=ATCC BAA-491 / LMD-9;
RX PubMed=22745249; DOI=10.1126/science.1225829;
RA Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E.;
RT "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial
RT immunity.";
RL Science 337:816-821(2012).
RN [3]
RP BIOTECHNOLOGY.
RC STRAIN=ATCC BAA-491 / LMD-9;
RX PubMed=23287718; DOI=10.1126/science.1231143;
RA Cong L., Ran F.A., Cox D., Lin S., Barretto R., Habib N., Hsu P.D., Wu X.,
RA Jiang W., Marraffini L.A., Zhang F.;
RT "Multiplex genome engineering using CRISPR/Cas systems.";
RL Science 339:819-823(2013).
RN [4]
RP FUNCTION AS AN ENDONUCLEASE, AND FUNCTION IN GUIDE RNA PROCESSING.
RC STRAIN=ATCC BAA-491 / LMD-9;
RX PubMed=24270795; DOI=10.1093/nar/gkt1074;
RA Fonfara I., Le Rhun A., Chylinski K., Makarova K.S., Lecrivain A.L.,
RA Bzdrenga J., Koonin E.V., Charpentier E.;
RT "Phylogeny of Cas9 determines functional exchangeability of dual-RNA and
RT Cas9 among orthologous type II CRISPR-Cas systems.";
RL Nucleic Acids Res. 42:2577-2590(2014).
RN [5]
RP FUNCTION, AND DOMAIN.
RX PubMed=24505130; DOI=10.1126/science.1247997;
RA Jinek M., Jiang F., Taylor D.W., Sternberg S.H., Kaya E., Ma E., Anders C.,
RA Hauer M., Zhou K., Lin S., Kaplan M., Iavarone A.T., Charpentier E.,
RA Nogales E., Doudna J.A.;
RT "Structures of Cas9 endonucleases reveal RNA-mediated conformational
RT activation.";
RL Science 343:1247997-1247997(2014).
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 (By similarity). The tracrRNA serves as a guide for
CC ribonuclease 3-aided processing of pre-crRNA. Subsequently
CC Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular
CC dsDNA target complementary to the spacer yielding blunt ends; Cas9 is
CC inactive in the absence of the 2 guide RNAs (gRNA). Cas9 recognizes a
CC 3'-G-rich protospacer adjacent motif (PAM, GGG in this organism) in the
CC CRISPR repeat sequences to help distinguish self versus nonself, as
CC targets within the bacterial CRISPR locus do not have PAMs. PAM
CC recognition is also required for catalytic activity. Complements the
CC gRNA coprocessing defect in a cas9 deletion in S.pyogenes strain 370,
CC and cuts target DNA in Cas9:gRNAs mixing experiments with S.mutans
CC strain UA159. {ECO:0000255|HAMAP-Rule:MF_01480,
CC ECO:0000269|PubMed:22745249, ECO:0000269|PubMed:24270795,
CC ECO:0000269|PubMed:24505130}.
CC -!- COFACTOR:
CC Name=Mg(2+); Xref=ChEBI:CHEBI:18420; Evidence={ECO:0000305};
CC Note=Endonuclease activity on target dsDNA requires Mg(2+).
CC {ECO:0000305};
CC -!- SUBUNIT: Monomer (By similarity). Binds crRNA and tracrRNA.
CC {ECO:0000255|HAMAP-Rule:MF_01480, ECO:0000269|PubMed:22745249}.
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 -!- DOMAIN: The PAM-interacting domain (PI domain, approximately residues
CC 1102-1388) recognizes the PAM motif; swapping the PI domain of this
CC enzyme with that from S.pyogenes Cas9 (AC Q99ZW2) prevents cleavage of
CC DNA with the endogenous PAM site but confers the ability to cleave DNA
CC with the PAM site specific for S.pyogenes CRISPRs.
CC {ECO:0000269|PubMed:24505130}.
CC -!- BIOTECHNOLOGY: Coexpression of Cas9 and both gRNAs in human cells has
CC shown it is possible to use this system to target and modify a DNA
CC sequence of interest in situ. {ECO:0000269|PubMed:23287718}.
CC -!- SIMILARITY: Belongs to the CRISPR-associated protein Cas9 family.
CC Subtype II-A subfamily. {ECO:0000255|HAMAP-Rule:MF_01480, ECO:0000305}.
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DR EMBL; CP000419; ABJ66636.1; -; Genomic_DNA.
DR RefSeq; WP_011681470.1; NC_008532.1.
DR AlphaFoldDB; Q03JI6; -.
DR SMR; Q03JI6; -.
DR PRIDE; Q03JI6; -.
DR KEGG; ste:STER_1477; -.
DR HOGENOM; CLU_005604_0_0_9; -.
DR OMA; TDRHSIK; -.
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..1388
FT /note="CRISPR-associated endonuclease Cas9 2"
FT /id="PRO_0000421686"
FT DOMAIN 771..928
FT /note="HNH Cas9-type"
FT /evidence="ECO:0000255|PROSITE-ProRule:PRU01085"
FT REGION 1100..1130
FT /note="Disordered"
FT /evidence="ECO:0000256|SAM:MobiDB-lite"
FT REGION 1102..1388
FT /note="PAM-interacting domain (PI)"
FT ACT_SITE 10
FT /note="For RuvC-like nuclease domain"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_01480"
FT ACT_SITE 847
FT /note="Proton acceptor for HNH nuclease domain"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_01480"
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 763
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /ligand_label="1"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_01480"
FT BINDING 767
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /ligand_label="1"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_01480"
FT BINDING 767
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /ligand_label="2"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_01480"
FT BINDING 990
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /ligand_label="2"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_01480"
SQ SEQUENCE 1388 AA; 161031 MW; B97BC4C953090235 CRC64;
MTKPYSIGLD IGTNSVGWAV TTDNYKVPSK KMKVLGNTSK KYIKKNLLGV LLFDSGITAE
GRRLKRTARR RYTRRRNRIL YLQEIFSTEM ATLDDAFFQR LDDSFLVPDD KRDSKYPIFG
NLVEEKAYHD EFPTIYHLRK YLADSTKKAD LRLVYLALAH MIKYRGHFLI EGEFNSKNND
IQKNFQDFLD TYNAIFESDL SLENSKQLEE IVKDKISKLE KKDRILKLFP GEKNSGIFSE
FLKLIVGNQA DFRKCFNLDE KASLHFSKES YDEDLETLLG YIGDDYSDVF LKAKKLYDAI
LLSGFLTVTD NETEAPLSSA MIKRYNEHKE DLALLKEYIR NISLKTYNEV FKDDTKNGYA
GYIDGKTNQE DFYVYLKKLL AEFEGADYFL EKIDREDFLR KQRTFDNGSI PYQIHLQEMR
AILDKQAKFY PFLAKNKERI EKILTFRIPY YVGPLARGNS DFAWSIRKRN EKITPWNFED
VIDKESSAEA FINRMTSFDL YLPEEKVLPK HSLLYETFNV YNELTKVRFI AESMRDYQFL
DSKQKKDIVR LYFKDKRKVT DKDIIEYLHA IYGYDGIELK GIEKQFNSSL STYHDLLNII
NDKEFLDDSS NEAIIEEIIH TLTIFEDREM IKQRLSKFEN IFDKSVLKKL SRRHYTGWGK
LSAKLINGIR DEKSGNTILD YLIDDGISNR NFMQLIHDDA LSFKKKIQKA QIIGDEDKGN
IKEVVKSLPG SPAIKKGILQ SIKIVDELVK VMGGRKPESI VVEMARENQY TNQGKSNSQQ
RLKRLEKSLK ELGSKILKEN IPAKLSKIDN NALQNDRLYL YYLQNGKDMY TGDDLDIDRL
SNYDIDHIIP QAFLKDNSID NKVLVSSASN RGKSDDVPSL EVVKKRKTFW YQLLKSKLIS
QRKFDNLTKA ERGGLSPEDK AGFIQRQLVE TRQITKHVAR LLDEKFNNKK DENNRAVRTV
KIITLKSTLV SQFRKDFELY KVREINDFHH AHDAYLNAVV ASALLKKYPK LEPEFVYGDY
PKYNSFRERK SATEKVYFYS NIMNIFKKSI SLADGRVIER PLIEVNEETG ESVWNKESDL
ATVRRVLSYP QVNVVKKVEE QNHGLDRGKP KGLFNANLSS KPKPNSNENL VGAKEYLDPK
KYGGYAGISN SFTVLVKGTI EKGAKKKITN VLEFQGISIL DRINYRKDKL NFLLEKGYKD
IELIIELPKY SLFELSDGSR RMLASILSTN NKRGEIHKGN QIFLSQKFVK LLYHAKRISN
TINENHRKYV ENHKKEFEEL FYYILEFNEN YVGAKKNGKL LNSAFQSWQN HSIDELCSSF
IGPTGSERKG LFELTSRGSA ADFEFLGVKI PRYRDYTPSS LLKDATLIHQ SVTGLYETRI
DLAKLGEG