Abstract
DNA base editors, typically comprising editing enzymes fused to the N-terminus of nCas9, display off-target effects on DNA and/or RNA, which have remained an obstacle to their clinical applications. Off-target edits are typically countered via rationally designed point mutations, but the approach is tedious and not always effective. Here, we report that the off-target effects of both A > G and C > T editors can be dramatically reduced without compromising the on-target editing simply by inserting the editing enzymes into the middle of nCas9 at tolerant sites identified using a transposon-based genetic screen. Furthermore, employing this Cas-embedding strategy, we have created a highly specific editor capable of efficient C > T editing at methylated and GC-rich sequences.
Highlights
AncBE4max consisting of the APOBEC ancestor Anc[689] linked to the N terminus of nCas[9], which is highly active but presumably highly non-specific[17]
The second CBE we sought to optimize involves BE4-A3A, comprising APOBEC3A fused to the N terminus of nCas[9]
For A3A editors, CE-mediated suppression of RNA off-target editing was much more pronounced, reducing the SNV numbers 100×, from 2025 in BE4max-A3A(Y130F) to only 18 in CE1048–1063-A3A(Y130F), a level only slightly above the GFP background (11) (Fig. 4d). These results show that the CE strategy is applicable to CBEs, enabling the creation of CE1048–1063-A3A(Y130F), a highly specific editor capable of robust editing at methylated or presumably GC-rich sequences
Summary
Primers and plasmids are listed in Supplementary Table 1. PCMV-nCas9-KanR-AmpR(A118X)-sgRNA, the all-in-one plasmid for insertion screening, was assembled from pCMV-ABEmax (Addgene 112095), pUC57-Kan (Addgene, 51132), and pGL3-U6-sgRNA (Addgene, 51133). The sgRNA expression vector for mammalian cells was constructed using BsaI-digested pGL3-U6sgRNA-EGFP with annealed DNA oligos (Supplementary Table 1). The sgRNA expression vector for GOTI was constructed by cloning annealed DNA oligos (Supplementary Table 1) into BbsI-digested pUC57-sgRNA (Addgene, 51132). Fragments encompassing the target sites (~200 bp) were PCR-amplified using Phanta Max Super-Fidelity DNA polymerase (Vazyme, P505-03); the primers used are listed in Supplementary Table 1. All the processed reads were mapped to the target sequences using the BWA-MEM algorithm (BWA v0.7.16). Indels were calculated based on the reads containing at least one inserted or deleted nucleotide in the protospacer. Indel frequency was expressed as the number of indelcontaining reads/total mapped reads
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