Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction
- Heidelberg University, Germany
- University of Oviedo, Spain
Abstract
Single nucleotide variants (SNVs) are prevalent genetic factors shaping individual trait profiles and disease susceptibility. The recent development and optimizations of base editors, rubber and pencil genome editing tools now promise to enable direct functional assessment of SNVs in model organisms. However, the lack of bioinformatic tools aiding target prediction limits the application of base editing in vivo. Here, we provide a framework for adenine and cytosine base editing in medaka (Oryzias latipes) and zebrafish (Danio rerio), ideal for scalable validation studies. We developed an online base editing tool ACEofBASEs (a careful evaluation of base-edits), to facilitate decision-making by streamlining sgRNA design and performing off-target evaluation. We used state-of-the-art adenine (ABE) and cytosine base editors (CBE) in medaka and zebrafish to edit eye pigmentation genes and transgenic GFP function with high efficiencies. Base editing in the genes encoding troponin T and the potassium channel ERG faithfully recreated known cardiac phenotypes. Deep-sequencing of alleles revealed the abundance of intended edits in comparison to low levels of insertion or deletion (indel) events for ABE8e and evoBE4max. We finally validated missense mutations in novel candidate genes of congenital heart disease (CHD) dapk3, ube2b, usp44, and ptpn11 in F0 and F1 for a subset of these target genes with genotype-phenotype correlation. This base editing framework applies to a wide range of SNV-susceptible traits accessible in fish, facilitating straight-forward candidate validation and prioritization for detailed mechanistic downstream studies.
Data availability
Source code for ACEofBASEs has been provided.
Article and author information
Author details
Funding
Deutsche Forschungsgemeinschaft (WI 1824/9-1)
- Joachim Wittbrodt
H2020 European Research Council (810172)
- Joachim Wittbrodt
Deutsches Zentrum für Herz-Kreislaufforschung
- Joachim Wittbrodt
Deutsche Herzstiftung (S/02/17)
- Jakob Gierten
Joachim Herz Stiftung
- Jakob Gierten
Deutsche Forschungsgemeinschaft (3DMM2O)
- Joachim Wittbrodt
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2022, Cornean et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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