Precise base editing for the in vivo study of developmental signaling and human pathologies in zebrafish

  1. Marion Rosello
  2. Juliette Vougny
  3. François Czarny
  4. Marina C Mione
  5. Jean-Paul Concordet
  6. Shahad Albadri  Is a corresponding author
  7. Filippo Del Bene  Is a corresponding author
  1. Sorbonne Université, INSERM, CNRS, Institut de la Vision, France
  2. Institut Curie, PSL Research University, Inserm U934, CNRS UMR3215, France
  3. Department of Cellular, Computational and Integrative Biology – CIBIO, University of Trento, Italy
  4. Muséum National d’Histoire Naturelle, INSERM U1154, CNRS UMR 7196, France
3 figures, 3 tables and 2 additional files

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’.

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 AD) or ancBE4max-SpymacCas9 (in E,F) and obtained C-to-T conversion efficiencies. (A) E62K mutation in Kras upon C-to-T …

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 embryos5/87/88/100/84/70/82/48/82/41/4
Highest obtained efficiency73%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 embryosEmb. 1Emb. 2Emb. 3Emb. 4Emb. 5Emb. 6Emb. 7Emb. 8Emb. 9Emb. 10
ctnnb1 (S33L)
BE4-gam
5/8C15
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
5/8C14
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
4/814%12%9%8%n.d.n.d.n.d.n.d.
tp53 (Q21*)
BE4-gam
6/863%33%37%58%8%50%n.d.n.d.
tp53 (Q170*)
BE4-gam
7/886%46%51%62%45%20%33%n.d.
cbl (W577*)
BE4-gam
8/10C16
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
0/8n.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.
kras
(E62K) ancBE4max
4/7C17
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
0/8n.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.
dmd
(Q8*) ancBE4max
2/414%6%n.d.n.d.
sod2 (Q145*) ancBE4max8/864%45%21%54%52%24%26%33%
rb1
(W63*) ancBE4max
8/8C19
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
2/419%18%n.d.n.d.
tp53 (Q170*) spymac-ancBE4max1/416%n.d.n.d.n.d.
Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional
information
Genetic reagent (Danio rerio)Tg(7xTCF-Xla.Siam:GFP)ZIRCZFIN ID:ZBD-ALT-110113–1
Recombinant DNA reagentpCMV_BE4-gam
(plasmid)
AddgeneAddgene:#100806
RRID:Addgene_100806
Recombinant DNA reagentpCMV_ancBE4max
(plasmid)
AddgeneAddgene:#112094
RRID:Addgene_112094
Recombinant DNA reagentpCS2+_ancBE4max-SpymacCas9
(plasmid)
This paperSee Materials and methods
Commercial assay or kitNEBuilder HiFi DNA Assembly Cloning KitNew England BiolabsCatalog# E5520S
Commercial assay or kitmMESSAGE mMACHINE T7 Ultra kitAmbionCatalog# AM1345
Commercial assay or kitmMESSAGE mMACHINE Sp6 kitAmbionCatalog# AM1340
Commercial assay or kitPCR clean-up gel extraction kitMacherey-NagelCatalog# 740609.50
Peptide, recombinant proteinPhusion high-fidelity DNA polymeraseThermoFisherCatalog# F-530XL
Software, algorithmSequenceParser.pyThis paperSee Source code 1

Additional files

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
Transparent reporting form
https://cdn.elifesciences.org/articles/65552/elife-65552-transrepform-v2.docx

Download links