(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 …
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’.
(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 …
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% |
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. | – | – | – | – | – | – |
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 |
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).