Programmable deaminase-free base editors for G-to-Y conversion by engineered glycosylase

Abstract

Current DNA base editors contain nuclease and DNA deaminase that enables deamination of cytosine (C) or adenine (A), but no method for guanine (G) or thymine (T) editing is available now. Here we developed a deaminase-free glycosylase-based guanine base editor (gGBE) with G editing ability, by fusing Cas9 nickase with engineered N-methylpurine DNA glycosylase protein (MPG). By several rounds of MPG mutagenesis via unbiased and rational screening using an intron-split EGFP reporter, we demonstrated that gGBE with engineered MPG could increase G editing efficiency by more than 1,500 folds. Furthermore, this gGBE exhibited high base editing efficiency (up to 81.2%) and high G-to-T or G-to-C (i.e., G-to-Y) conversion ratio (up to 0.95) in both cultured human cells and mouse embryos. Thus, we have provided a proof-of-concept of a new base-editing approach by endowing the engineered DNA glycosylase the capability to selectively excising a new type of substrate.

Development of deaminase-free T-to-S base editor and C-to-G base editor by engineered human uracil DNA glycosylase

“…3a and Supplementary Fig. 6i), consistent with previous findings of gGBE 3 , AYBE 9 and CGBEs 11–15 . We found that gTBEv3 also induced indels with frequency ranging from 5.2% to 45.2% at the 20 edited sites (Fig.…”

“…Encouraged by the development of gGBE in our previous study 3 , we attempted to develop thymine and cytosine base editor using the deaminase-free glycosylase-based strategy. Since the three pyrimidine bases (i.e., T, C, and U) are structurally similar, we speculated that excision of canonical T or C could be achieved by engineering certain uracil DNA glycosylase, thus triggering the BER pathway and facilitating direct T editing or C editing following the one-step generation of AP sites (Fig.…”

“…Base editors enable single-nucleotide edits with high precision and efficiency, providing powerful tools for the fields of life science and medicine 1, 2 . Two categories of DNA base editors, deaminase-based base editor (dBE) and deaminase-free glycosylase-based base editor (gBE), have been developed to date 3 . The dBEs, including adenine base editor (ABE) 4 , cytosine base editor (CBE) 5 , and double-stranded DNA deaminase toxin A (DddA)-derived cytosine base editor (DdCBE) 6, 7 as well as their derivatives (such as A&C-BEmax 8 , AYBE 9 , AXBE 10 and CGBEs 11–15 ), perform base editing using single-stranded or double-stranded DNA deaminase enzymes (e.g., evolved tRNA adenosine deaminase TadA, AID/APOBEC-like cytidine deaminase or DddA variants).…”

“…All the base conversions by dBEs require the deamination of A or C as the first key step. Recently, we have developed a gBE enabling direct G editing (i.e., deaminase-free glycosylase-based guanine base editor, gGBE) 3 , based on engineered human N-methylpurine DNA glycosylase (MPG; also known as alkyladenine DNA glycosylase, AAG). So far, dBEs and gGBE could enable editing of adenine (A), cytosine (C), or guanine (G), but no base editor for thymine (T) editing is available now.…”

Design and Engineering of Light‐Induced Base Editors Facilitating Genome Editing with Enhanced Fidelity

“…Additionally, we showed that the rifampin assay, which was used to identify suitable split sites, was feasible for cytosine deaminases as well as for adenine deaminases. Thus, this strategy can be applied to other deaminases, such as APOBEC3B, [ 59 ] TadA orthologs, [ 70 ] and even the non‐deaminase N‐methylpurine DNA glycosylase, [ 71 ] which has been shown to achieve G‐to‐Y editing.…”