Jul 30, 2020

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

Posted by in categories: biotech/medical, evolution

CRISPR-Cas9 base editors comprise RNA-guided Cas proteins fused to an enzyme that can deaminate a DNA nucleoside. No natural enzyme deaminates adenine in DNA, and so a breakthrough came when a natural transfer RNA deaminase was fused to Cas9 and evolved to give an adenine base editor (ABE) that works on DNA. Further evolution provided the enzyme ABE8e, which catalyzes deamination more than 1000 times faster than early ABEs. Lapinaite et al. now present a 3.2-angstrom resolution structure of ABE8e bound to DNA in which the target adenine is replaced with an analog designed to trap the catalytic conformation. The structure, together with kinetic data comparing ABE8e to earlier ABEs, explains how ABE8e edits DNA bases and could inform future base-editor design.

Science, this issue p. 566

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.

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