Adenine Base Editor Ribonucleoproteins Delivered by Lentivirus-Like Particles Show High On-Target Base Editing and Undetectable RNA Off-Target Activities

CRISPR J. 2021 Feb;4(1):69-81. doi: 10.1089/crispr.2020.0095.

Abstract

Adenine base editors (ABEs) can correct gene mutations without creating double-strand breaks. However, in recent reports, these editors showed guide-independent RNA off-target activities. This work describes our development of a delivery method to minimize ABEs' RNA off-target activity. After discovering a RNA off-target hot spot for sensitive detection of RNA off-target activities, we found that delivering ribonucleoproteins (RNPs) by electroporation generated undetectable non-specific RNA editing, but on-target base editing activity was also relatively low. We then explored a lentivirus capsid-based delivery strategy to deliver ABE. We used aptamer/aptamer-binding protein (ABP) interactions to package ABE RNPs into lentiviral capsids. Capsid RNPs were delivered to human cells for highly efficient guided base editing. Importantly, RNA off-target activities from the capsid RNPs were undetectable. Our new lentiviral capsid-based ABE RNP delivery method with minimal RNA off-target activities makes ABE one step closer to possible therapeutic applications.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adenine / metabolism*
  • Aptamers, Nucleotide
  • Base Sequence
  • Capsid / metabolism
  • Gene Editing / methods*
  • HEK293 Cells
  • Humans
  • Lentivirus / genetics*
  • Mutation
  • RNA
  • RNA Editing*
  • Ribonucleoproteins / genetics*

Substances

  • Aptamers, Nucleotide
  • Ribonucleoproteins
  • RNA
  • Adenine