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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Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction
eLife 11:e72124.
https://doi.org/10.7554/eLife.72124
Further reading
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- Developmental Biology
Congenital malformations can originate from numerous genetic or non-genetic factors but in most cases the causes are unknown. Genetic disruption of nicotinamide adenine dinucleotide (NAD) de novo synthesis causes multiple malformations, collectively termed Congenital NAD Deficiency Disorder (CNDD), highlighting the necessity of this pathway during embryogenesis. Previous work in mice shows that NAD deficiency perturbs embryonic development specifically when organs are forming. While the pathway is predominantly active in the liver postnatally, the site of activity prior to and during organogenesis is unknown. Here, we used a mouse model of human CNDD and assessed pathway functionality in embryonic livers and extraembryonic tissues via gene expression, enzyme activity and metabolic analyses. We found that the extra-embryonic visceral yolk sac endoderm exclusively synthesises NAD de novo during early organogenesis before the embryonic liver takes over this function. Under CNDD-inducing conditions, visceral yolk sacs had reduced NAD levels and altered NAD-related metabolic profiles, affecting embryo metabolism. Expression of requisite pathway genes is conserved in the equivalent yolk sac cell type in humans. Our findings show that visceral yolk sac-mediated NAD de novo synthesis activity is essential for mouse embryogenesis and its perturbation causes CNDD. As mouse and human yolk sacs are functionally homologous, our data improve the understanding of human congenital malformation causation.
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- Developmental Biology
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