Expanding the Editing Window of Cytidine Base Editors With the Rad51 DNA-Binding Domain in Rice

Abstract

Recently developed base editors provide a powerful tool for plant research and crop improvement. Although a number of different deaminases and Cas proteins have been used to improve base editors the editing efficiency, and editing window are still not optimal. Fusion of a non-sequence-specific single-stranded DNA-binding domain (DBD) from the human Rad51 protein between Cas9 nickase and the deaminase has been reported to dramatically increase the editing efficiency and expand the editing window of base editors in the mammalian cell lines and mouse embryos. We report the use of this strategy in rice, by fusing a rice codon-optimized human Rad51 DBD to the cytidine base editors AncBE4max, AncBE4max-NG, and evoFERNY. Our results show that the addition of Rad51 DBD did not increase editing efficiency in the major editing window but the editing range was expanded in all the three systems. Replacing the human Rad51 DBD with the rice Rad51 DBD homolog also expanded the editing window effectively.

Gene editing and scalable functional genomic screening in Leishmania species using the CRISPR/Cas9 cytosine base editor toolbox LeishBASEedit

“…We show that our Leishmania -optimised hyBE4max CBE version increases the editing efficiency ( Figure 7B and Figure 7—figure supplement 1C ). However, it remains to be explored whether also fidelity and editing window size is affected by this modification, as for example hyBE4max adaptations in rise are known to have expanded the editing windows ( Wei et al, 2022 ). While especially our improved CBE version allowed to efficiently induce a knockdown of CRK3, guides targeting IFT88 were not significantly depleted in our small-scale library screen ( Figure 7B ).…”

Introducing the CRISPR/Cas9 cytosine base editor toolbox ‘LeishBASEedit’ – Gene editing and high-throughput screening inLeishmaniawithout requiring DNA double-strand breaks, homologous recombination or donor DNA

“…We show that our Leishmania -optimised hyBE4max CBE version increases the editing efficiency (Figure 7B and S11C). However, it remains to be explored whether also fidelity and editing window size is affected by this modification, as for example hyBE4max adaptations in rise are known to have expanded the editing windows (77). While especially our improved CBE version allowed to efficiently induce a knockdown of CRK3, guides targeting IFT88 were not significantly depleted in our small-scale library screen (Figure 7B).…”

Design and characterization of multiplex gene-edited CAR-T cells resistant to orthogonal immunosuppressive pathways within the solid tumor microenvironment

“…Base editors have a more restricted targeting capacity, owing to their precise editing of a single nucleotide, which may hinder the design of these newer therapeutics requiring editing far beyond a single KO. New base editors containing different PAMs, extended editing windows, or enhanced catalytic activity may solve this potential problem [143][144][145]. Extending base editor target accessibility to match that of traditional CRISPR-Cas9 may be required for full therapeutic capacity of this platform.…”