Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction
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...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2022-04-01
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| Series: | eLife |
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| Online Access: | https://elifesciences.org/articles/72124 |
| _version_ | 1811252280128700416 |
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| author | Alex Cornean Jakob Gierten Bettina Welz Juan Luis Mateo Thomas Thumberger Joachim Wittbrodt |
| author_facet | Alex Cornean Jakob Gierten Bettina Welz Juan Luis Mateo Thomas Thumberger Joachim Wittbrodt |
| author_sort | Alex Cornean |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-12T16:32:27Z |
| format | Article |
| id | doaj.art-f07c3ffac51544a2a599d72722eb004d |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-12T16:32:27Z |
| publishDate | 2022-04-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-f07c3ffac51544a2a599d72722eb004d2022-12-22T03:25:06ZengeLife Sciences Publications LtdeLife2050-084X2022-04-011110.7554/eLife.72124Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target predictionAlex Cornean0https://orcid.org/0000-0003-3727-7057Jakob Gierten1https://orcid.org/0000-0001-8143-1918Bettina Welz2Juan Luis Mateo3https://orcid.org/0000-0001-9902-6048Thomas Thumberger4https://orcid.org/0000-0001-8485-457XJoachim Wittbrodt5https://orcid.org/0000-0001-8550-7377Centre for Organismal Studies, Heidelberg University, Heidelberg, Germany; Heidelberg Biosciences International Graduate School (HBIGS), Heidelberg, GermanyCentre for Organismal Studies, Heidelberg University, Heidelberg, Germany; Department of Pediatric Cardiology, University Hospital Heidelberg, Heidelberg, Germany; DZHK (German Centre for Cardiovascular Research), Heidelberg, GermanyCentre for Organismal Studies, Heidelberg University, Heidelberg, Germany; Heidelberg Biosciences International Graduate School (HBIGS), Heidelberg, Germany; DZHK (German Centre for Cardiovascular Research), Heidelberg, GermanyDeparment of Computer Science, University of Oviedo, Oviedo, SpainCentre for Organismal Studies, Heidelberg University, Heidelberg, GermanyCentre for Organismal Studies, Heidelberg University, Heidelberg, Germany; DZHK (German Centre for Cardiovascular Research), Heidelberg, GermanySingle 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.https://elifesciences.org/articles/72124medakabase editingdisease variant validationSNV |
| spellingShingle | Alex Cornean Jakob Gierten Bettina Welz Juan Luis Mateo Thomas Thumberger Joachim Wittbrodt Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction eLife medaka base editing disease variant validation SNV |
| title | Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction |
| title_full | Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction |
| title_fullStr | Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction |
| title_full_unstemmed | Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction |
| title_short | Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction |
| title_sort | precise in vivo functional analysis of dna variants with base editing using aceofbases target prediction |
| topic | medaka base editing disease variant validation SNV |
| url | https://elifesciences.org/articles/72124 |
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