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- EMDB-35035: Cryo-EM structure of the EvCas9-sgRNA-target DNA ternary complex -
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Open data
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Basic information
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Title | Cryo-EM structure of the EvCas9-sgRNA-target DNA ternary complex | |||||||||
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![]() | RNA / DNA / RNA BINDING PROTEIN/RNA/DNA / RNA BINDING PROTEIN-RNA-DNA complex | |||||||||
Function / homology | ![]() maintenance of CRISPR repeat elements / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding Similarity search - Function | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.43 Å | |||||||||
![]() | Tang N / Wu Z / Gao Y / Chen W / Su M / Wang Z / Ji Q | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Molecular Basis and Genome Editing Applications of a Compact CRISPR-Cas9 System. Authors: Na Tang / Zhaowei Wu / Yan Gao / Weizhong Chen / Zixiao Wang / Mengjiao Su / Wenxin Ji / Quanjiang Ji / ![]() Abstract: CRISPR-Cas9 systems have been widely harnessed for diverse genome editing applications because of their ease of use and high efficiency. However, the large molecular sizes and strict PAM requirements ...CRISPR-Cas9 systems have been widely harnessed for diverse genome editing applications because of their ease of use and high efficiency. However, the large molecular sizes and strict PAM requirements of commonly used CRISPR-Cas9 systems restrict their broad applications in therapeutics. Here, we report the molecular basis and genome editing applications of a novel compact type II-A CRISPR-Cas9 system (EvCas9) with 1107 residues and distinct 5'-NNGDGN-3' (where D represents A, T, or G) PAM specificity. We determine the cryo-EM structure of EvCas9 in a complex with an sgRNA and a target DNA, revealing the detailed PAM recognition and sgRNA and target DNA association mechanisms. Additionally, we demonstrate the robust genome editing capacity of EvCas9 in bacteria and human cells with superior fidelity compared to SaCas9 and SpCas9, and we engineer it to be efficient base editors by fusing a cytidine or adenosine deaminase. Collectively, our results facilitate further understanding of CRISPR-Cas9 working mechanisms and expand the compact CRISPR-Cas9 toolbox. | |||||||||
History |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 203.8 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 18.8 KB 18.8 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 14.3 KB | Display | ![]() |
Images | ![]() | 101.5 KB | ||
Filedesc metadata | ![]() | 6.7 KB | ||
Others | ![]() ![]() | 200.5 MB 200.5 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8hudMC M: atomic model generated by this map C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Voxel size | X=Y=Z: 0.82 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #1
File | emd_35035_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_35035_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
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Sample components
-Entire : Ternary complex of EvCas9-sgRNA-dsDNA
Entire | Name: Ternary complex of EvCas9-sgRNA-dsDNA |
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Components |
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-Supramolecule #1: Ternary complex of EvCas9-sgRNA-dsDNA
Supramolecule | Name: Ternary complex of EvCas9-sgRNA-dsDNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 156.5 KDa |
-Macromolecule #1: CRISPR-associated endonuclease Cas9
Macromolecule | Name: CRISPR-associated endonuclease Cas9 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 131.110531 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MGYTVGLDIG VASVGVAVLD ENDNIVEAVS NIFDEADTSN NKVRRTLREG RRTKRRQKTR IEDFKQLWET SGYIIPHKLH LNIIELRNK GLTELLSLDE LYCVLLSMLK HRGISYLEDA DDGEKGNAYK KGLAFNEKQL KEKMPCEIQL ERMKKYGKYH G EFIIEIND ...String: MGYTVGLDIG VASVGVAVLD ENDNIVEAVS NIFDEADTSN NKVRRTLREG RRTKRRQKTR IEDFKQLWET SGYIIPHKLH LNIIELRNK GLTELLSLDE LYCVLLSMLK HRGISYLEDA DDGEKGNAYK KGLAFNEKQL KEKMPCEIQL ERMKKYGKYH G EFIIEIND EKEYQSNVFT TKAYKKELEK IFETQRCNGN KINTKFIKKY MEIYERKREY YIGPGNEKSR TDYGIYTTRT DE EGNFIDE KNIFGKLIGK CSVYPEEYRA SSASYTAQEF NLLNDLNNLK INNEKLTEFQ KKEIVEIIKD ASSVNMRKII KKV IDEDIE QYSGARIDKK GKEIYHTFEI YRKLKKELKT INVDIDSFTR EELDKTMDIL TLNTERESIV KAFDEQKFVY EENL IKKLI EFRKNNQRLF SGWHSFSYKA MLQLIPVMYK EPKEQMQLLT EMNVFKSKKE KYVNYKYIPE NEVVKEIYNP VVVKS IRTT VKILNALIKK YGYPESVVIE MPRDKNSDDE KEKIDMNQKK NQEEYEKILN KIYDEKGIEI TNKDYKKQKK LVLKLK LWN EQEGLCLYSG KKIAIEDLLN HPEFFEIDHI IPKSISLDDS RSNKVLVYKT ENSIKENDTP YHYLTRINGK WGFDEYK AN VLELRRRGKI DDKKVNNLLC MEDITKIDVV KGFINRNLND TRYASRVVLN EMQSFFESRK YCNTKVKVIR GSLTYQMR Q DLHLKKNREE SYSHHAVDAM LIAFSQKGYE AYRKIQKDCY DFETGEILDK EKWNKYIDDD EFDDILYKER MNEIRKKII EAEEKVKYNY KIDKKCNRGL CNQTIYGTRE KDGKIHKISS YNIYDDKECN SLKKMINSGK GSDLLMYNND PKTYRDMLKI LETYSSEKN PFVAYNKETG DYFRKYSKNH NGPKVEKVKY YSGQINSCID ISHKYGHAKN SKKVVLVSLN PYRTDVYYDN D TGKYYLVG VKYNHIKCVG NKYVIDSETY NELLRKEGVL NSDENLEDLN SKNITYKFSL YKNDIIQYEK GGEYYTERFL SR IKEQKNL IETKPINKPN FQRKNKKGEW ENTRNQIALA KTKYVGKLVT DVLGNCYIVN MEKFSLVVDK UniProtKB: CRISPR-associated endonuclease Cas9 |
-Macromolecule #2: sgRNA
Macromolecule | Name: sgRNA / type: rna / ID: 2 / Number of copies: 1 |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 23.97624 KDa |
Sequence | String: GGUAAUCGCU CUCCUCCGGC GAUUUUAGUA CCUGAGAAAU CAGAUCUACU AAAACAAGGC UUUAUGCCGA AAUCA |
-Macromolecule #3: Target DNA strand
Macromolecule | Name: Target DNA strand / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 8.641558 KDa |
Sequence | String: (DC)(DG)(DC)(DC)(DA)(DC)(DG)(DT)(DC)(DG) (DC)(DC)(DG)(DG)(DA)(DG)(DG)(DA)(DG)(DA) (DG)(DC)(DG)(DA)(DT)(DT)(DA)(DC) |
-Macromolecule #4: Non-target DNA strand
Macromolecule | Name: Non-target DNA strand / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 2.467629 KDa |
Sequence | String: (DA)(DC)(DG)(DT)(DG)(DG)(DC)(DG) |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.5 mg/mL | |||||||||||||||
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Buffer | pH: 7.5 Component:
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Grid | Model: Homemade / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: OTHER | |||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281.15 K / Instrument: FEI VITROBOT MARK IV |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 16.8 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 1.2 µm |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |