DNA capture by a CRISPR-Cas9-guided adenine base editor

Audrone 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

doi:10.1126/science.abb1390

DNA capture by a CRISPR-Cas9-guided adenine base editor

Science. 2020 Jul 31;369(6503):566-571. doi: 10.1126/science.abb1390.

Authors

Audrone Lapinaite^#¹, Gavin J Knott^#^{1

2}, Cody M Palumbo³, Enrique Lin-Shiao¹, Michelle F Richter^{4

5

6}, Kevin T Zhao^{4

5

6}, Peter A Beal³, David R Liu^{4

5

6}, Jennifer A Doudna^{7

8

9

10

11

12}

Affiliations

¹ Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
² Monash Biomedicine Discovery Institute, Department of Biochemistry & Molecular Biology, Monash University, Victoria 3800, Australia.
³ Department of Chemistry, University of California, Davis, CA 95616, USA.
⁴ Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA 02141, USA.
⁵ Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
⁶ Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA.
⁷ Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA. doudna@berkeley.edu.
⁸ Innovative Genomics Institute, University of California, Berkeley, CA 94720, USA.
⁹ MBIB Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
¹⁰ Department of Chemistry, University of California, Berkeley, CA 94720, USA.
¹¹ Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA.
¹² Gladstone Institute of Data Science and Biotechnology, Gladstone Institutes, San Francisco, CA 94158, USA.

^# Contributed equally.

Abstract

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.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Adenine / chemistry*
Adenosine Deaminase / chemistry*
Adenosine Deaminase / genetics
CRISPR-Associated Protein 9 / chemistry*
CRISPR-Associated Protein 9 / genetics
CRISPR-Cas Systems*
Cryoelectron Microscopy
DNA / chemistry*
Deamination
Escherichia coli Proteins / chemistry*
Escherichia coli Proteins / genetics
Gene Editing*

Substances

Escherichia coli Proteins
DNA
CRISPR-Associated Protein 9
Adenosine Deaminase
TadA protein, E coli
Adenine

Abstract

Publication types

MeSH terms

Substances

Grants and funding