Precise base editing for the in vivo study of developmental signaling and human pathologies in zebrafish
- Sorbonne Université, INSERM, CNRS, Institut de la Vision, France
- Institut Curie, PSL Research University, Inserm U934, CNRS UMR3215, France
- Department of Cellular, Computational and Integrative Biology – CIBIO, University of Trento, Italy
- Muséum National d’Histoire Naturelle, INSERM U1154, CNRS UMR 7196, France
Figures
Figure 1 with 1 supplement
Efficient endogenous activation of Wnt signaling pathway and tumor suppressor genes targeting using BE4-gam in zebrafish.
(A) Schematic representation of the cytidine base editor technology. (B) Activation of Wnt signaling via S33L mutation in β-catenin. 1 dpf Tg(7xTCF-Xla.Siam:GFP) representative embryos injected with …
Figure 1—figure supplement 1
List of targeted loci.
List of all the targeted loci in this study. In red are highlighted the targeted C bases, underlined are the sgRNAs and in green the associated PAM sequences. Sequences are oriented from 5’ to 3’.
Figure 2
Tumor suppressor genes and oncogenes targeting by the highly efficient ancBE4max and the ancBE4max-SpymacCas9 recognizing NAA PAM.
(A–F) DNA sequencing chromatogram of targeted loci with the ancBE4max (in A–D) or ancBE4max-SpymacCas9 (in E,F) and obtained C-to-T conversion efficiencies. (A) E62K mutation in Kras upon C-to-T …
Figure 3
BE4-gam generated cbl maternal zygotic mutant fish show a reduced growth phenotype.
(A) DNA sequencing chromatogram of targeted cbl gene with the BE4-gam. W577* mutation in Cbl upon C-to-T conversion in cbl reached 50% for the C16 base and 35% for the C15 base of gene-editing …
Tables
Table 1
Base-editing efficiency using different CBE variants.
Number of edited embryos randomly chosen after injection of CBE mRNA and sgRNA. The efficiency varies between non-detected (n.d.) and 91% depending on the targeted locus, the sgRNA, and the CBE …
| Targeted gene CBE used induced mutation | ctnnb1 (S33L) BE4-gam | tp53 (Q170*) BE4-gam | cbl (W577*) BE4-gam | kras (E62K) BE4-gam | Kras (E62K) ancBE4max | dmd (Q8*) BE4-gam | dmd (Q8*) ancBE4max | rb1 (W63*) ancBE4max | nras (G13S) spymac -ancBE4max | tp53 (Q170*) spymac- ancBE4max | ||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Number of edited embryos | 5/8 | 7/8 | 8/10 | 0/8 | 4/7 | 0/8 | 2/4 | 8/8 | 2/4 | 1/4 | ||
| Highest obtained efficiency | 73% | 86% | C16 35% | C15 50% | n.d. | 19% | n.d. | 14% | C17 91% | C16 65% | 19% | 16% |
Table 2
Editing efficiency quantification.
Editing quantification of up to 10 single embryos randomly chosen after injection of indicated CBE mRNA and sgRNA. The efficiency varies between non-detected (n.d.) to 91% in a single embryo …
| Targeted gene CBE used | Number of edited embryos | Emb. 1 | Emb. 2 | Emb. 3 | Emb. 4 | Emb. 5 | Emb. 6 | Emb. 7 | Emb. 8 | Emb. 9 | Emb. 10 | ||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ctnnb1 (S33L) BE4-gam | 5/8 | C15 74% | C13 73% | C15 n.d. | C13 40% | C15 44% | C13 25% | C15 7% | C13 16% | C15 n.d. | C13 11% | n.d. | n.d. | n.d. | – | – | |||||||||||
| tek (Q94*) BE4-gam | 5/8 | C14 18% | C13 8% | C14 10% | C13 n.d. | C14 8% | C13 n.d. | C14 6% | C13 n.d. | C14 8% | C13 9% | n.d. | n.d. | n.d. | – | – | |||||||||||
| Bap1 (Q273*) BE4-gam | 4/8 | 14% | 12% | 9% | 8% | n.d. | n.d. | n.d. | n.d. | – | – | ||||||||||||||||
| tp53 (Q21*) BE4-gam | 6/8 | 63% | 33% | 37% | 58% | 8% | 50% | n.d. | n.d. | – | – | ||||||||||||||||
| tp53 (Q170*) BE4-gam | 7/8 | 86% | 46% | 51% | 62% | 45% | 20% | 33% | n.d. | – | – | ||||||||||||||||
| cbl (W577*) BE4-gam | 8/10 | C16 35% | C15 50% | C16 19% | C15 31% | C16 22% | C15 38% | C16 25% | C15 41% | C16 20% | C15 35% | C16 7% | C15 9% | C16 7% | C15 12% | C16 10% | C15 17% | n.d. | n.d. | ||||||||
| kras (E62K) BE4-gam | 0/8 | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | – | – | ||||||||||||||||
| kras (E62K) ancBE4max | 4/7 | C17 19% | C16 21% | C17 8% | C16 11% | C17 6% | C16 8% | C17 9% | C16 10% | n.d. | n.d. | n.d. | – | – | – | ||||||||||||
| dmd (Q8*) BE4-gam | 0/8 | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | – | – | ||||||||||||||||
| dmd (Q8*) ancBE4max | 2/4 | 14% | 6% | n.d. | n.d. | – | – | – | – | – | – | ||||||||||||||||
| sod2 (Q145*) ancBE4max | 8/8 | 64% | 45% | 21% | 54% | 52% | 24% | 26% | 33% | – | – | ||||||||||||||||
| rb1 (W63*) ancBE4max | 8/8 | C19 n.d. | C17 91% | C16 65% | C19 21% | C17 79% | C16 75% | C19 n.d. | C17 27% | C16 18% | C19 13% | C17 81% | C16 60% | C19 8% | C17 48% | C16 33% | C19 13% | C17 76% | C16 64% | C19 13% | C17 78% | C16 69% | C19 21% | C17 77% | C16 63% | – | – |
| nras (G13S) spymac-ancBE4max | 2/4 | 19% | 18% | n.d. | n.d. | – | – | – | – | – | – | ||||||||||||||||
| tp53 (Q170*) spymac-ancBE4max | 1/4 | 16% | n.d. | n.d. | n.d. | – | – | – | – | – | – | ||||||||||||||||
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Danio rerio) | Tg(7xTCF-Xla.Siam:GFP) | ZIRC | ZFIN ID:ZBD-ALT-110113–1 | |
| Recombinant DNA reagent | pCMV_BE4-gam (plasmid) | Addgene | Addgene:#100806 RRID:Addgene_100806 | |
| Recombinant DNA reagent | pCMV_ancBE4max (plasmid) | Addgene | Addgene:#112094 RRID:Addgene_112094 | |
| Recombinant DNA reagent | pCS2+_ancBE4max-SpymacCas9 (plasmid) | This paper | See Materials and methods | |
| Commercial assay or kit | NEBuilder HiFi DNA Assembly Cloning Kit | New England Biolabs | Catalog# E5520S | |
| Commercial assay or kit | mMESSAGE mMACHINE T7 Ultra kit | Ambion | Catalog# AM1345 | |
| Commercial assay or kit | mMESSAGE mMACHINE Sp6 kit | Ambion | Catalog# AM1340 | |
| Commercial assay or kit | PCR clean-up gel extraction kit | Macherey-Nagel | Catalog# 740609.50 | |
| Peptide, recombinant protein | Phusion high-fidelity DNA polymerase | ThermoFisher | Catalog# F-530XL | |
| Software, algorithm | SequenceParser.py | This paper | See Source code 1 |
Additional files
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Source code 1
SequenceParser.py STOP codon design source code.
This python code highlights in capital the codons that can converted as STOP codon by C-to-T conversion with the chosen PAM sequence at the correct distance (PAM [−19, –13] bp window).
- https://cdn.elifesciences.org/articles/65552/elife-65552-code1-v2.zip
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Transparent reporting form
- https://cdn.elifesciences.org/articles/65552/elife-65552-transrepform-v2.docx
