Abstract
Precision genome engineering has dramatically advanced with the development of CRISPR/Cas base editing systems that include cytosine base editors and adenine base editors (ABEs). Herein, we compare the editing profile of circularly permuted and domain-inlaid Cas9 base editors, and find that on-target editing is largely maintained following their intradomain insertion, but that structural permutation of the ABE can affect differing RNA off-target events. With this insight, structure-guided design was used to engineer an SaCas9 ABE variant (microABE I744) that has dramatically improved on-target editing efficiency and a reduced RNA-off target footprint compared to current N-terminal linked SaCas9 ABE variants. This represents one of the smallest AAV-deliverable Cas9-ABEs available, which has been optimized for robust on-target activity and RNA-fidelity based upon its stereochemistry.
Highlights
Precision genome engineering has dramatically advanced with the development of CRISPR/ Cas base editing systems that include cytosine base editors and adenine base editors (ABEs)
Circular permutant variants of the hAIDx base editor at nSpCas[9] residues 1010, 1029, and 1058 (Supplementary Fig. 1) were selected for a direct comparison of on-target editing using a cell line expressing yellow fluorescent protein (YFP), which has no homologous analog in the human genome
We show that the same variant of ABE can have different DNA and RNA editing profiles arising from alterations to their secondary structure
Summary
Precision genome engineering has dramatically advanced with the development of CRISPR/ Cas base editing systems that include cytosine base editors and adenine base editors (ABEs). Current generation adenine base editors (ABEs) employ a dimerized, codon optimized variant of laboratory-evolved ecTadA (ABEmax)[4,5], and have directed site-specific adenine-to-guanine nucleotide conversions in a diverse array of systems[6,7] Despite their broad scope for robust on-target editing, non-engineered ABEs have a significant off-target footprint on the transcriptome and effect incidences of missense and nonsense mutations[8]. Efforts to minimize the occurrence of promiscuous editing have largely improved the fidelity of existing ABEs by installing various inactivating mutations in the wild-type domain of the ecTadA monomer[9], or use truncated variants of ABEmax with amino acid substitution to reduce non-specific contacts with RNA in the recently described, miniABEmax, which consists of a single, evolved ecTadA monomer[10,11]. By fine-tuning the spatial positioning between the base editor and Cas[9] component, this work represents a useful addendum to efforts enhancing base editing activity and fidelity
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