BE4max and AncBE4max Are Efficient in Germline Conversion of C:G to T:A Base Pairs in Zebrafish

Carrington, Blake; Weinstein, Rachel N.

doi:10.3390/cells9071690

Cited by 21 publications

(14 citation statements)

References 58 publications

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“…In comparison, previous work has reported an efficiency reaching a maximum of 29% using the BE3 ( Zhang et al, 2017 ). Another more recent study employed the ancBE4max variant in zebrafish with a slight difference of efficiency that might be due to the choice of the specific locus targeted, the synthesis of the sgRNA (homemade vs commercially synthetized) and the injection mode (yolk vs cell) ( Carrington et al, 2020 ). More recently, Zhao et al, 2020 have shown similar efficiencies as we obtained in our study.…”

Section: Resultsmentioning

confidence: 99%

“…CBE converts C-to-T bases in a restricted window of 13–19 nucleotides (nt) upstream of the PAM sequence ( Figure 1A ). In zebrafish, a CBE was shown to work but with limited efficiencies, inducing less than 29% of gene editing and, in most cases, at least 5% of unwanted INDEL (insertion or deletion) mutations were also detected ( Carrington et al, 2020 ; Zhang et al, 2017 ). For these reasons, this editing strategy has not been so far favored by the zebrafish community.…”

Section: Introductionmentioning

confidence: 99%

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

Rosello

Vougny

Czarny

et al. 2021

eLife

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While zebrafish is emerging as a new model system to study human diseases, an efficient methodology to generate precise point mutations at high efficiency is still lacking. Here we show that base editors can generate C-to-T point mutations with high efficiencies without other unwanted on-target mutations. In addition, we established a new editor variant recognizing an NAA PAM, expanding the base editing possibilities in zebrafish. Using these approaches, we first generated a base change in the ctnnb1 gene, mimicking oncogenic mutations of the human gene known to result in constitutive activation of endogenous Wnt signaling. Additionally, we precisely targeted several cancer-associated genes including cbl. With this last target we created a new zebrafish dwarfism model. Together our findings expand the potential of zebrafish as a model system allowing new approaches for the endogenous modulation of cell signaling pathways and the generation of precise models of human genetic disease associated-mutations.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Rosello

Vougny

Czarny

et al. 2021

eLife

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show abstract

“…1A). In zebrafish, a CBE has been shown to work but efficiencies were limited, with less than 29% editing being induced and, in most cases, at least 5% of unwanted INDEL (Insertion or Deletion) mutations were detected (Carrington et al, 2020;Zhang et al, 2017). For these reasons, these editing strategies have not been so far favored by the zebrafish community and no direct CBE applications have been performed to the study of developmental processes or to generate human disease models.…”

Section: Introductionmentioning

confidence: 99%

Precise base editing for thein vivostudy of developmental signaling and human pathologies in zebrafish

Rosello

Vougny

Czarny

et al. 2020

Preprint

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While zebrafish is emerging as a new model system to study human diseases, an efficient methodology to generate precise point mutations at high efficiency is still lacking. Here we show that base editors can generate C-to-T point mutations with high efficiencies without other unwanted on-target mutations. In addition, we established a new editor variant recognizing an NAA PAM, expanding the base editing possibilities in zebrafish. Using these approaches, we first generated a base change in the ctnnb1 gene, mimicking oncogenic mutations of the human gene known to result in constitutive activation of endogenous Wnt signaling. Additionally, we precisely targeted several cancer-associated genes among which cbl. With this last target we created a new zebrafish dwarfism model. Together our findings expand the potential of zebrafish as a model system allowing new approaches for the endogenous modulation of cell signaling pathways and the generation of precise models of human genetic disease associated-mutations.

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“…With slight modifications this system has been successfully used in zebrafish [ 169 ]. The CRISPR “base editing” toolkit is constantly being improved, creating new possibilities for editing [ 170 , 171 ].…”

Section: Animal Models For Drug Screening Using Genetically Encodementioning

confidence: 99%

Drug Screening with Genetically Encoded Fluorescent Sensors: Today and Tomorrow

Potekhina

Bass

Kelmanson

et al. 2020

IJMS

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Genetically-encoded fluorescent sensors have been actively developed over the last few decades and used in live imaging and drug screening. Real-time monitoring of drug action in a specific cellular compartment, organ, or tissue type; the ability to screen at the single-cell resolution; and the elimination of false-positive results caused by low drug bioavailability that is not detected by in vitro testing methods are a few of the obvious benefits of using genetically-encoded fluorescent sensors in drug screening. In combination with high-throughput screening (HTS), some genetically-encoded fluorescent sensors may provide high reproducibility and robustness to assays. We provide a brief overview of successful, perspective, and hopeful attempts at using genetically encoded fluorescent sensors in HTS of modulators of ion channels, Ca2+ homeostasis, GPCR activity, and for screening cytotoxic, anticancer, and anti-parasitic compounds. We discuss the advantages of sensors in whole organism drug screening models and the perspectives of the combination of human disease modeling by CRISPR techniques with genetically encoded fluorescent sensors for drug screening.

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BE4max and AncBE4max Are Efficient in Germline Conversion of C:G to T:A Base Pairs in Zebrafish

Cited by 21 publications

References 58 publications

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

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

Precise base editing for thein vivostudy of developmental signaling and human pathologies in zebrafish

Drug Screening with Genetically Encoded Fluorescent Sensors: Today and Tomorrow

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