Highly reliable creation of floxed alleles by electroporating single-cell embryos

Monica F. Sentmanat; J. Michael White; Evguenia Kouranova; Xiaoxia Cui

doi:10.1186/s12915-021-01223-w

Highly reliable creation of floxed alleles by electroporating single-cell embryos

Monica F. Sentmanat, J. Michael White, Evguenia Kouranova, Xiaoxia Cui

Research output: Contribution to journal › Article › peer-review

8 Scopus citations

Abstract

Background: Floxed (flanked by loxP) alleles are a crucial portion of conditional knockout mouse models. However, an efficient and reliable strategy to flox genomic regions of any desired size is still lacking. Results: Here, we demonstrate that the method combining electroporation of fertilized eggs with gRNA/Cas9 complexes and single-stranded oligodeoxynucleotides (ssODNs), assessing phasing of loxP insertions in founders using an in vitro Cre assay and an optional, highly specific and efficient second-round targeting ensures the generation of floxed F1 animals in roughly five months for a wide range of sequence lengths (448 bp to 160 kb reported here). Conclusions: Floxed alleles can be reliably obtained in a predictable timeline using the improved method of electroporation of two gRNA/Cas9 ribonucleoprotein particles (RNPs) and two ssODNs.

Original language	English
Article number	31
Journal	BMC Biology
Volume	20
Issue number	1
DOIs	https://doi.org/10.1186/s12915-021-01223-w
State	Published - Dec 2022

Keywords

CRISPR
Conditional knockout mice
Floxing
Functional genomics
Gene editing
Mouse models

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10.1186/s12915-021-01223-w

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title = "Highly reliable creation of floxed alleles by electroporating single-cell embryos",

abstract = "Background: Floxed (flanked by loxP) alleles are a crucial portion of conditional knockout mouse models. However, an efficient and reliable strategy to flox genomic regions of any desired size is still lacking. Results: Here, we demonstrate that the method combining electroporation of fertilized eggs with gRNA/Cas9 complexes and single-stranded oligodeoxynucleotides (ssODNs), assessing phasing of loxP insertions in founders using an in vitro Cre assay and an optional, highly specific and efficient second-round targeting ensures the generation of floxed F1 animals in roughly five months for a wide range of sequence lengths (448 bp to 160 kb reported here). Conclusions: Floxed alleles can be reliably obtained in a predictable timeline using the improved method of electroporation of two gRNA/Cas9 ribonucleoprotein particles (RNPs) and two ssODNs.",

keywords = "CRISPR, Conditional knockout mice, Floxing, Functional genomics, Gene editing, Mouse models",

author = "Sentmanat, {Monica F.} and White, {J. Michael} and Evguenia Kouranova and Xiaoxia Cui",

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AU - Sentmanat, Monica F.

AU - White, J. Michael

AU - Kouranova, Evguenia

AU - Cui, Xiaoxia

PY - 2022/12

Y1 - 2022/12

N2 - Background: Floxed (flanked by loxP) alleles are a crucial portion of conditional knockout mouse models. However, an efficient and reliable strategy to flox genomic regions of any desired size is still lacking. Results: Here, we demonstrate that the method combining electroporation of fertilized eggs with gRNA/Cas9 complexes and single-stranded oligodeoxynucleotides (ssODNs), assessing phasing of loxP insertions in founders using an in vitro Cre assay and an optional, highly specific and efficient second-round targeting ensures the generation of floxed F1 animals in roughly five months for a wide range of sequence lengths (448 bp to 160 kb reported here). Conclusions: Floxed alleles can be reliably obtained in a predictable timeline using the improved method of electroporation of two gRNA/Cas9 ribonucleoprotein particles (RNPs) and two ssODNs.

AB - Background: Floxed (flanked by loxP) alleles are a crucial portion of conditional knockout mouse models. However, an efficient and reliable strategy to flox genomic regions of any desired size is still lacking. Results: Here, we demonstrate that the method combining electroporation of fertilized eggs with gRNA/Cas9 complexes and single-stranded oligodeoxynucleotides (ssODNs), assessing phasing of loxP insertions in founders using an in vitro Cre assay and an optional, highly specific and efficient second-round targeting ensures the generation of floxed F1 animals in roughly five months for a wide range of sequence lengths (448 bp to 160 kb reported here). Conclusions: Floxed alleles can be reliably obtained in a predictable timeline using the improved method of electroporation of two gRNA/Cas9 ribonucleoprotein particles (RNPs) and two ssODNs.

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KW - Conditional knockout mice

KW - Floxing

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KW - Gene editing

KW - Mouse models

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Highly reliable creation of floxed alleles by electroporating single-cell embryos

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Keywords

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