Secrets of a Fast Base Editor
CRISPR-Cas9 base editors include Cas R-guided proteins fused with an enzyme that can design a DNA nucleoside. No natural enzyme designs adenine in DNA, so a breakthrough was made when natural transfer RNA deaminase was fused to Cas9 and evolved to give a working adenine base (ABE) editor. on DNA. Further evolution provided the ABE8e enzyme, which catalyzes deamination more than 1000 times faster than early ABE. Lapinaite et al. I am now presenting a DNA-bound 3.2 angstrom resolution structure of ABE8e in which the targeted adenine is replaced by an analog designed to capture catalytic conformation. The structure, along with kinetic data comparing ABE8e with previous ABE, explains how ABE8e edits DNA bases and can inform future design of a base editor.
Science, this issue p. 566
CRISPR-Cas-guided base editors convert A • T to G • C, or C • G to T • A, into cellular DNA for precision genome editing. To understand the molecular basis for adenosine DNA deamination from adenine-based editors (ABEs), we determined a cryo-electron microscopy structure with a 3.2 angstrom resolution of ABE8e in a bound state. with the substrate in which the deaminase domain enters exposed DNA within the CRISPR-Cas9 R-line complex. Kinetic and structural data suggest that ABE8e catalyzes DNA deamination up to ~ 1100 times faster than previous ABE due to mutations which stabilize DNA substrates in a constrained, transferring conformation such as RNA. In addition, rapid DNA deamination of ABE8e suggests previously unobserved transient DNA melting that may occur during two-line DNA monitoring by CRISPR-Cas9. These results explain the base editing outcomes mediated by ABE8e and inform the future design of the base editors.