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Structure paper

TitleDNA capture by a CRISPR-Cas9-guided adenine base editor.
Journal, issue, pagesScience, Vol. 369, Issue 6503, Page 566-571, Year 2020
Publish dateJul 31, 2020
AuthorsAudrone Lapinaite / Gavin J Knott / Cody M Palumbo / Enrique Lin-Shiao / Michelle F Richter / Kevin T Zhao / Peter A Beal / David R Liu / Jennifer A Doudna /
PubMed AbstractCRISPR-Cas-guided base editors convert A•T to G•C, or C•G to T•A, in cellular DNA for precision genome editing. To understand the molecular basis for DNA adenosine deamination by adenine base ...CRISPR-Cas-guided base editors convert A•T to G•C, or C•G to T•A, in cellular DNA for precision genome editing. To understand the molecular basis for DNA adenosine deamination by adenine base editors (ABEs), we determined a 3.2-angstrom resolution cryo-electron microscopy structure of ABE8e in a substrate-bound state in which the deaminase domain engages DNA exposed within the CRISPR-Cas9 R-loop complex. Kinetic and structural data suggest that ABE8e catalyzes DNA deamination up to ~1100-fold faster than earlier ABEs because of mutations that stabilize DNA substrates in a constrained, transfer RNA-like conformation. Furthermore, ABE8e's accelerated DNA deamination suggests a previously unobserved transient DNA melting that may occur during double-stranded DNA surveillance by CRISPR-Cas9. These results explain ABE8e-mediated base-editing outcomes and inform the future design of base editors.
External linksScience / PubMed:32732424 / PubMed Central
MethodsEM (single particle)
Resolution3.2 Å
Structure data

21308

6vpc

EMDB-21308, PDB-6vpc:
Structure of the SpCas9 DNA adenine base editor - ABE8e
Method: EM (single particle) / Resolution: 3.2 Å

Chemicals

ChemComp-MG:
Unknown entry

ChemComp-ZN:
Unknown entry

Source
  • streptococcus pyogenes (bacteria)
  • escherichia coli (E. coli)
  • synthetic construct (others)
KeywordsDNA BINDING PROTEIN/DNA/RNA / Base editor / ABE / Cas9 / CRISPR / DNA BINDING PROTEIN-DNA-RNA complex

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