Eliminating predictable DNA off-target effects of cytosine base editor by using dual guiders including sgRNA and TALE

Mol Ther. 2022 Jul 6;30(7):2443-2451. doi: 10.1016/j.ymthe.2022.04.010. Epub 2022 Apr 20.

Abstract

Predictable DNA off-target effect is one of the major safety concerns for the application of cytosine base editors (CBEs). To eliminate Cas9-dependent DNA off-target effects, we designed a novel effective CBE system with dual guiders by combining CRISPR with transcription activator-like effector (TALE). In this system, Cas9 nickase (nCas9) and cytosine deaminase are guided to the same target site to conduct base editing by single-guide RNA (sgRNA) and TALE, respectively. However, if nCas9 is guided to a wrong site by sgRNA, it will not generate base editing due to the absence of deaminase. Similarly, when deaminase is guided to a wrong site by TALE, base editing will not occur due to the absence of single-stranded DNA. In this way, Cas9- and TALE-dependent DNA off-target effects could be completely eliminated. Furthermore, by fusing TALE with YE1, a cytidine deaminase with minimal Cas9-independent off-target effect, we established a novel CBE that could induce efficient C-to-T conversion without detectable Cas9- or TALE-dependent DNA off-target mutations.

Keywords: CRISPR-Cas9; TALE; cytosine base editor; predictable DNA off-target effects.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems
  • Cytosine*
  • DNA / genetics
  • Gene Editing
  • RNA, Guide, CRISPR-Cas Systems* / genetics
  • Transcription Activator-Like Effectors / genetics

Substances

  • RNA, Guide, CRISPR-Cas Systems
  • Transcription Activator-Like Effectors
  • Cytosine
  • DNA