Skip Header

You are using a version of browser that may not display all the features of this website. Please consider upgrading your browser.
Protein

CRISPR-associated endonuclease Cas12a

Gene

cas12a

Organism
Francisella tularensis subsp. novicida (strain U112)
Status
Reviewed-Annotation score: Annotation score: 5 out of 5-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). CRISPR clusters contain sequences complementary to antecedent mobile elements and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). Has endonuclease activity on pre-crRNA and dsDNA, using different active sites. A single-RNA guided endonuclease that is also capable of guiding crRNA processing; correct processing of pre-crRNA requires only this protein and the CRISPR locus (PubMed:26422227, PubMed:27096362). pre-crRNA processing proceeds by an intramolecular nucleophilic attack on the scissile phosphate by the 2'-OH of the upstream ribonucleotide, the divalent cation (which is bound by the crRNA) is probably required for ordering the crRNA pseudoknot and/or increasing RNA binding (PubMed:28431230). RNA mutagenesis studies show pre-crRNA cleavage is highly sequence- and structure-specific (PubMed:27096362). Forms a complex with crRNA and complementary dsDNA, where the crRNA displaces the non-target DNA strand and directs endonucleolytic cleavage of both strands of the DNA (PubMed:26422227, PubMed:27096362, PubMed:28431230). Cleavage results in staggered 5-base 5' overhangs 14-18 and 21-23 bases downstream of the PAM (protospacer adjacent motif) on the non-target and target strands respectively (PubMed:26422227, PubMed:28431230, PubMed:28562584). Both target and non-target strand DNA are probably independently cleaved in the same active site (PubMed:28431230, PubMed:28562584). When this protein is expressed in E.coli it prevents plasmids homologous to the first CRISPR spacer from transforming, formally showing it is responsible for plasmid immunity (PubMed:26422227).4 Publications

Miscellaneous

Part of a type V-A CRISPR-Cas system.2 Publications

Catalytic activityi

Endonucleolytic cleavage to 5'-phosphodinucleotide and 5'-phosphooligonucleotide end-products.1 Publication
Endonucleolytic cleavage to 2',3'-cyclic nucleotides.1 Publication

Cofactori

Ca2+1 Publication, Mg2+4 PublicationsNote: Cleavage of dsDNA requires Mg2+ (PubMed:26422227, PubMed:28562584). Another report shows DNA cleavage occurs equally well in the presence of Ca2+ or Mg2+ (PubMed:27096362). Processing of pre-crRNA requires a divalent cation, preferably Mg2+ which is bound by the crRNA (PubMed:26593719, PubMed:28431230).5 Publications

Sites

Feature keyPosition(s)DescriptionActionsGraphical viewLength
Binding sitei16crRNA alone and in crRNA-target DNA heteroduplex2 Publications1
Binding sitei131Target strand DNA1 Publication1
Binding sitei295crRNA in crRNA-target DNA heteroduplex1 Publication1
Binding sitei320DNA in crRNA-target DNA heteroduplex1 Publication1
Binding sitei334DNA in crRNA-target DNA heteroduplex1 Publication1
Sitei410Caps the crRNA-target DNA heteroduplex1 Publication1
Binding sitei589DNA in crRNA-target DNA heteroduplex1 Publication1
Binding sitei613DNA protospacer adjacent motif (PAM)2 Publications1
Binding sitei667Target strand DNA1 Publication1
Binding sitei671PAM2 Publications1
Binding sitei677Target strand DNA2 Publications1
Binding sitei823Target strand DNA2 Publications1
Binding sitei826Target strand DNA; via amide nitrogen2 Publications1
Binding sitei833crRNA1 Publication1
Active sitei843For pre-crRNA processing1 Publication1
Active sitei852For pre-crRNA processing1 Publication1
Sitei852Stabilizes transition state for pre-crRNA processing1 Publication1
Active sitei869For pre-crRNA processing1 Publication1
Active sitei917For DNase activity of RuvC domain1 Publication1
Active sitei1006For DNase activity of RuvC domain1 Publication1
Binding sitei1026DNA in crRNA-target DNA heteroduplex2 Publications1
Binding sitei1063DNA in crRNA-target DNA heteroduplex2 Publications1
Active sitei1255For DNase activity of RuvC domain1 Publication1

GO - Molecular functioni

GO - Biological processi

Keywordsi

Molecular functionDNA-binding, Endonuclease, Hydrolase, Nuclease, RNA-binding
Biological processAntiviral defense
LigandCalcium, Magnesium

Enzyme and pathway databases

BioCyciFTUL401614:G12WZ-1387-MONOMER.

Names & Taxonomyi

Protein namesi
Recommended name:
CRISPR-associated endonuclease Cas12a1 Publication (EC:3.1.21.11 Publication, EC:3.1.27.21 Publication)
Alternative name(s):
CRISPR-associated endonuclease Cpf11 Publication
FnCas12a1 Publication
FnCpf11 Publication
Gene namesi
Name:cas12a1 Publication
Synonyms:cpf11 Publication
Ordered Locus Names:FTN_1397
OrganismiFrancisella tularensis subsp. novicida (strain U112)
Taxonomic identifieri401614 [NCBI]
Taxonomic lineageiBacteriaProteobacteriaGammaproteobacteriaThiotrichalesFrancisellaceaeFrancisella
Proteomesi
  • UP000000762 Componenti: Chromosome

Pathology & Biotechi

Biotechnological usei

This class of CRISPR enzymes recognize a 5' T-rich protospacer adjacent motif (PAM, TTN for this specific enzyme), unlike Cas9 enzymes which recognize 3' G-rich PAMs, thus this enzyme increases the possibilites for genome editing (PubMed:26422227). The simplicity of the Cas12a-crRNA directed DNA endonuclease activity has been used to target and modify DNA sequences in rice and tobacco (PubMed:27905529).1 Publication2 Publications

Mutagenesis

Feature keyPosition(s)DescriptionActionsGraphical viewLength
Mutagenesisi608G → A or E: 15% DNA cleavage. 1 Publication1
Mutagenesisi663P → A: 25% DNA cleavage, altered non-target strand cleavage products. 1 Publication1
Mutagenesisi666N → A: 80% DNA cleavage, altered non-target strand cleavage products. 1 Publication1
Mutagenesisi667K → A: 30% DNA cleavage. 1 Publication1
Mutagenesisi671K → A: 15% DNA cleavage. 1 Publication1
Mutagenesisi677K → A: 35% DNA cleavage, altered non-target strand cleavage products. 1 Publication1
Mutagenesisi692R → A: Slight decrease in target DNA cleavage, 30% DNA cleavage, altered non-target strand cleavage products. 2 Publications1
Mutagenesisi694H → A: Wild-type DNA cleavage, altered non-target strand cleavage products. 1 Publication1
Mutagenesisi698 – 702TKNGS → AGGGG: Loss of target DNA cleavage. 1 Publication5
Mutagenesisi704Q → A: Significant decrease in target DNA cleavage. 1 Publication1
Mutagenesisi843H → A: Decreased pre-crRNA processing in vitro, binds RNA, no change in DNA cleavage. 1 Publication1
Mutagenesisi852K → A: Decreased pre-crRNA processing in vitro, binds RNA, no change in DNA cleavage. 1 Publication1
Mutagenesisi869K → A: Decreased pre-crRNA processing in vitro, binds RNA, no change in DNA cleavage. 1 Publication1
Mutagenesisi873F → A: Decreased pre-crRNA processing in vitro, no pre-crRNA processing in E.coli, binds RNA, no change in DNA cleavage. 1 Publication1
Mutagenesisi917D → A: Loss of target and non-target strand DNA cleavage, no change in DNA-binding or pre-crRNA processing. 3 Publications1
Mutagenesisi920E → A: No longer cleaves DNA in presence of Ca(2+). 1 Publication1
Mutagenesisi922H → A: Decreased cleavage of target strand in presence of Ca(2+), wild-type cleavage of DNA in presence of Mg(2+). 1 Publication1
Mutagenesisi925Y → A: Decreased cleavage of target strand in presence of Ca(2+), wild-type cleavage of DNA in presence of Mg(2+). 1 Publication1
Mutagenesisi1006E → A: Loss of target and non-target strand DNA cleavage, no change in DNA-binding or pre-crRNA processing. 4 Publications1
Mutagenesisi1006E → Q: Complete loss of DNA cleavage, still binds crRNA; when associated with A-1218. 1 Publication1
Mutagenesisi1024Y → A: No longer cleaves DNA in presence of Ca(2+). 1 Publication1
Mutagenesisi1028E → A: No longer cleaves DNA in presence of Ca(2+), reduced cleavage of non-target strand in presence of Mg(2+). 1 Publication1
Mutagenesisi1065 – 1066KK → AA: 67% DNA cleavage, altered non-target strand cleavage products. 1 Publication2
Mutagenesisi1218R → A: Cleaves both target and non-target strand DNA. Complete loss of DNA cleavage, still binds crRNA; when associated with Q-1006. 2 Publications1
Mutagenesisi1227D → A: No longer cleaves DNA in presence of Ca(2+). 1 Publication1
Mutagenesisi1255D → A: Significant reduction to loss of target and non-target strand DNA cleavage, no change in DNA-binding or pre-crRNA processing. 3 Publications1
Mutagenesisi1255D → N: Significant reduction of target and non-target strand DNA cleavage. 1 Publication1

PTM / Processingi

Molecule processing

Feature keyPosition(s)DescriptionActionsGraphical viewLength
ChainiPRO_00004349021 – 1300CRISPR-associated endonuclease Cas12aAdd BLAST1300

Interactioni

Subunit structurei

Might be a homodimer (PubMed:26422227). Might be a monomer (PubMed:27096362, PubMed:28431230).3 Publications

Structurei

Secondary structure

11300
Legend: HelixTurnBeta strandPDB Structure known for this area
Show more details
Feature keyPosition(s)DescriptionActionsGraphical viewLength
Turni3 – 6Combined sources4
Beta strandi13 – 23Combined sources11
Helixi27 – 34Combined sources8
Helixi36 – 68Combined sources33
Helixi73 – 86Combined sources14
Helixi92 – 114Combined sources23
Helixi117 – 120Combined sources4
Beta strandi122 – 124Combined sources3
Helixi125 – 127Combined sources3
Beta strandi132 – 134Combined sources3
Helixi137 – 147Combined sources11
Helixi153 – 155Combined sources3
Helixi162 – 171Combined sources10
Turni172 – 174Combined sources3
Helixi176 – 179Combined sources4
Helixi180 – 190Combined sources11
Beta strandi192 – 194Combined sources3
Beta strandi196 – 198Combined sources3
Helixi199 – 204Combined sources6
Helixi207 – 224Combined sources18
Helixi226 – 228Combined sources3
Helixi231 – 237Combined sources7
Turni238 – 242Combined sources5
Beta strandi243 – 245Combined sources3
Turni248 – 251Combined sources4
Beta strandi252 – 257Combined sources6
Helixi260 – 263Combined sources4
Helixi267 – 271Combined sources5
Beta strandi272 – 274Combined sources3
Helixi275 – 286Combined sources12
Beta strandi291 – 293Combined sources3
Beta strandi294 – 297Combined sources4
Helixi300 – 311Combined sources12
Helixi314 – 319Combined sources6
Helixi345 – 361Combined sources17
Helixi370 – 382Combined sources13
Helixi388 – 390Combined sources3
Beta strandi392 – 394Combined sources3
Helixi397 – 406Combined sources10
Helixi412 – 422Combined sources11
Helixi438 – 442Combined sources5
Turni445 – 447Combined sources3
Beta strandi449 – 452Combined sources4
Helixi453 – 465Combined sources13
Beta strandi469 – 471Combined sources3
Helixi475 – 484Combined sources10
Helixi487 – 506Combined sources20
Helixi513 – 515Combined sources3
Helixi517 – 519Combined sources3
Helixi520 – 541Combined sources22
Helixi559 – 572Combined sources14
Helixi575 – 586Combined sources12
Beta strandi595 – 597Combined sources3
Turni603 – 606Combined sources4
Helixi611 – 613Combined sources3
Helixi614 – 617Combined sources4
Beta strandi619 – 624Combined sources6
Beta strandi627 – 633Combined sources7
Helixi635 – 637Combined sources3
Turni638 – 641Combined sources4
Helixi643 – 648Combined sources6
Beta strandi650 – 661Combined sources12
Helixi665 – 673Combined sources9
Turni676 – 678Combined sources3
Helixi679 – 682Combined sources4
Helixi686 – 694Combined sources9
Turni695 – 697Combined sources3
Beta strandi706 – 708Combined sources3
Helixi714 – 730Combined sources17
Helixi734 – 737Combined sources4
Helixi744 – 746Combined sources3
Helixi750 – 760Combined sources11
Beta strandi761 – 769Combined sources9
Helixi771 – 779Combined sources9
Beta strandi782 – 789Combined sources8
Helixi791 – 793Combined sources3
Helixi803 – 812Combined sources10
Helixi814 – 818Combined sources5
Beta strandi821 – 824Combined sources4
Beta strandi829 – 833Combined sources5
Beta strandi852 – 856Combined sources5
Beta strandi857 – 861Combined sources5
Helixi871 – 874Combined sources4
Beta strandi877 – 887Combined sources11
Helixi896 – 906Combined sources11
Helixi908 – 910Combined sources3
Beta strandi912 – 917Combined sources6
Beta strandi923 – 929Combined sources7
Beta strandi935 – 944Combined sources10
Beta strandi950 – 952Combined sources3
Helixi953 – 968Combined sources16
Turni969 – 971Combined sources3
Helixi977 – 999Combined sources23
Beta strandi1001 – 1006Combined sources6
Beta strandi1008 – 1011Combined sources4
Helixi1019 – 1036Combined sources18
Beta strandi1041 – 1043Combined sources3
Beta strandi1045 – 1048Combined sources4
Beta strandi1050 – 1053Combined sources4
Helixi1065 – 1067Combined sources3
Beta strandi1069 – 1071Combined sources3
Beta strandi1074 – 1077Combined sources4
Beta strandi1083 – 1085Combined sources3
Turni1087 – 1089Combined sources3
Helixi1102 – 1110Combined sources9
Beta strandi1112 – 1118Combined sources7
Turni1119 – 1122Combined sources4
Beta strandi1123 – 1129Combined sources7
Helixi1130 – 1132Combined sources3
Beta strandi1141 – 1145Combined sources5
Beta strandi1150 – 1153Combined sources4
Beta strandi1165 – 1168Combined sources4
Helixi1170 – 1180Combined sources11
Helixi1192 – 1197Combined sources6
Helixi1201 – 1214Combined sources14
Beta strandi1218 – 1221Combined sources4
Turni1222 – 1225Combined sources4
Beta strandi1228 – 1230Combined sources3
Turni1236 – 1238Combined sources3
Beta strandi1242 – 1245Combined sources4
Helixi1254 – 1275Combined sources22
Beta strandi1278 – 1280Combined sources3
Helixi1288 – 1297Combined sources10

3D structure databases

Select the link destinations:
PDBei
RCSB PDBi
PDBji
Links Updated
PDB entryMethodResolution (Å)ChainPositionsPDBsum
5MGAX-ray3.00A1-1300[»]
5NFVX-ray2.50A2-1300[»]
5NG6X-ray3.34A/C/E/G2-1300[»]
ProteinModelPortaliA0Q7Q2.
SMRiA0Q7Q2.
ModBaseiSearch...
MobiDBiSearch...

Family & Domainsi

Region

Feature keyPosition(s)DescriptionActionsGraphical viewLength
Regioni1 – 24Wedge region 12 PublicationsAdd BLAST24
Regioni25 – 339Recognition domain 12 PublicationsAdd BLAST315
Regioni47 – 51Binds crRNA alone and in crRNA-target DNA heteroduplex2 Publications5
Regioni182 – 186Binds crRNA alone and in crRNA-target DNA heteroduplex2 Publications5
Regioni301 – 305Binds DNA in crRNA-target DNA heteroduplex1 Publication5
Regioni326 – 329Binds crRNA in crRNA-target DNA heteroduplex2 Publications4
Regioni340 – 591Recognition domain 22 PublicationsAdd BLAST252
Regioni538 – 541Binds crRNA in crRNA-target DNA heteroduplex1 Publication4
Regioni591 – 595Binds crRNA1 Publication5
Regioni592 – 662Wedge region 22 PublicationsAdd BLAST71
Regioni662 – 679LKL, important for PAM recognition and DNA unwinding1 PublicationAdd BLAST18
Regioni663 – 762PAM-interacting domain (PI)2 PublicationsAdd BLAST100
Regioni671 – 677Binds DNA protospacer adjacent motif (PAM) on target DNA2 Publications7
Regioni692 – 704Binds single-strand non-target DNA1 PublicationAdd BLAST13
Regioni763 – 892Wedge region 32 PublicationsAdd BLAST130
Regioni791 – 794Binds crRNA1 Publication4
Regioni803 – 804Binds crRNA1 Publication2
Regioni851 – 853Binds crRNA2 Publications3
Regioni865 – 873Binds crRNA2 Publications9
Regioni893 – 953RuvC-I2 PublicationsAdd BLAST61
Regioni954 – 971Bridge helix2 PublicationsAdd BLAST18
Regioni972 – 1078RuvC-II2 PublicationsAdd BLAST107
Regioni1079 – 1254Nuclease domain2 PublicationsAdd BLAST176
Regioni1255 – 1300RuvC-III2 PublicationsAdd BLAST46

Domaini

Has bilobed structure, with the REC lobe (residues 25-591) connected to the NUC lobe (662-1300) by a discontinuous wedge domain (PubMed:28431230, PubMed:28562584). The REC lobe binds the (pre-)crRNA and the crRNA-target DNA heteroduplex (PubMed:28431230, PubMed:28562584). The heteroduplex as well as part of the DNA downstream of the heteroduplex is protected in the central cavity formed by the NUC and REC lobes, which also positions target and non-target DNA for cleavage after domain rearrangement (PubMed:28431230, PubMed:28562584). The LKL region (residues 662 to 679) inserts into target dsDNA initiating its disruption to allow crRNA hybridization, is also involved in determining the non-target strand cleavage site (PubMed:28562584). A 'septum' formed by resdiues 197-204 and 1061-1070 separates the 2 DNA strands, preventing their reannealing, this region also influences the non-target cleavage site (PubMed:28562584).2 Publications

Sequence similaritiesi

Belongs to the CRISPR-associated endonuclease Cas12a family.1 Publication

Phylogenomic databases

HOGENOMiHOG000066230.
OMAiYLFQIYN.

Family and domain databases

InterProiView protein in InterPro
IPR027620. Cas12a.
TIGRFAMsiTIGR04330. cas_Cpf1. 1 hit.

Sequencei

Sequence statusi: Complete.

A0Q7Q2-1 [UniParc]FASTAAdd to basket

« Hide

        10         20         30         40         50
MSIYQEFVNK YSLSKTLRFE LIPQGKTLEN IKARGLILDD EKRAKDYKKA
60 70 80 90 100
KQIIDKYHQF FIEEILSSVC ISEDLLQNYS DVYFKLKKSD DDNLQKDFKS
110 120 130 140 150
AKDTIKKQIS EYIKDSEKFK NLFNQNLIDA KKGQESDLIL WLKQSKDNGI
160 170 180 190 200
ELFKANSDIT DIDEALEIIK SFKGWTTYFK GFHENRKNVY SSNDIPTSII
210 220 230 240 250
YRIVDDNLPK FLENKAKYES LKDKAPEAIN YEQIKKDLAE ELTFDIDYKT
260 270 280 290 300
SEVNQRVFSL DEVFEIANFN NYLNQSGITK FNTIIGGKFV NGENTKRKGI
310 320 330 340 350
NEYINLYSQQ INDKTLKKYK MSVLFKQILS DTESKSFVID KLEDDSDVVT
360 370 380 390 400
TMQSFYEQIA AFKTVEEKSI KETLSLLFDD LKAQKLDLSK IYFKNDKSLT
410 420 430 440 450
DLSQQVFDDY SVIGTAVLEY ITQQIAPKNL DNPSKKEQEL IAKKTEKAKY
460 470 480 490 500
LSLETIKLAL EEFNKHRDID KQCRFEEILA NFAAIPMIFD EIAQNKDNLA
510 520 530 540 550
QISIKYQNQG KKDLLQASAE DDVKAIKDLL DQTNNLLHKL KIFHISQSED
560 570 580 590 600
KANILDKDEH FYLVFEECYF ELANIVPLYN KIRNYITQKP YSDEKFKLNF
610 620 630 640 650
ENSTLANGWD KNKEPDNTAI LFIKDDKYYL GVMNKKNNKI FDDKAIKENK
660 670 680 690 700
GEGYKKIVYK LLPGANKMLP KVFFSAKSIK FYNPSEDILR IRNHSTHTKN
710 720 730 740 750
GSPQKGYEKF EFNIEDCRKF IDFYKQSISK HPEWKDFGFR FSDTQRYNSI
760 770 780 790 800
DEFYREVENQ GYKLTFENIS ESYIDSVVNQ GKLYLFQIYN KDFSAYSKGR
810 820 830 840 850
PNLHTLYWKA LFDERNLQDV VYKLNGEAEL FYRKQSIPKK ITHPAKEAIA
860 870 880 890 900
NKNKDNPKKE SVFEYDLIKD KRFTEDKFFF HCPITINFKS SGANKFNDEI
910 920 930 940 950
NLLLKEKAND VHILSIDRGE RHLAYYTLVD GKGNIIKQDT FNIIGNDRMK
960 970 980 990 1000
TNYHDKLAAI EKDRDSARKD WKKINNIKEM KEGYLSQVVH EIAKLVIEYN
1010 1020 1030 1040 1050
AIVVFEDLNF GFKRGRFKVE KQVYQKLEKM LIEKLNYLVF KDNEFDKTGG
1060 1070 1080 1090 1100
VLRAYQLTAP FETFKKMGKQ TGIIYYVPAG FTSKICPVTG FVNQLYPKYE
1110 1120 1130 1140 1150
SVSKSQEFFS KFDKICYNLD KGYFEFSFDY KNFGDKAAKG KWTIASFGSR
1160 1170 1180 1190 1200
LINFRNSDKN HNWDTREVYP TKELEKLLKD YSIEYGHGEC IKAAICGESD
1210 1220 1230 1240 1250
KKFFAKLTSV LNTILQMRNS KTGTELDYLI SPVADVNGNF FDSRQAPKNM
1260 1270 1280 1290 1300
PQDADANGAY HIGLKGLMLL GRIKNNQEGK KLNLVIKNEE YFEFVQNRNN
Length:1,300
Mass (Da):151,915
Last modified:January 9, 2007 - v1
Checksum:i601E903DE68C80DE
GO

Sequence databases

Select the link destinations:
EMBLi
GenBanki
DDBJi
Links Updated
CP000439 Genomic DNA. Translation: ABK90267.1.
RefSeqiWP_003040289.1. NZ_CP009633.1.

Genome annotation databases

EnsemblBacteriaiABK90267; ABK90267; FTN_1397.
KEGGiftn:FTN_1397.
ftx:AW25_605.

Similar proteinsi

Entry informationi

Entry nameiCS12A_FRATN
AccessioniPrimary (citable) accession number: A0Q7Q2
Entry historyiIntegrated into UniProtKB/Swiss-Prot: December 9, 2015
Last sequence update: January 9, 2007
Last modified: January 31, 2018
This is version 49 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. PDB cross-references
    Index of Protein Data Bank (PDB) cross-references
  2. SIMILARITY comments
    Index of protein domains and families