What question did this study set out to answer?

To establish a standardized, highly efficient protocol for generating large-fragment knock-in mouse models by delivering CRISPR gene-editing components into two-cell-stage embryos.

January 8, 2020Current Protocols in Mouse Biology

Efficient Generation of Large‐Fragment Knock‐In Mouse Models Using 2‐Cell (2C)‐Homologous Recombination (HR)‐CRISPR

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Authors

BGBin GuSoochow University EPEszter PósfaiPrinceton University MGMarina GertsensteinToronto Centre for Phenogenomics

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Overview

Experimental study demonstrates efficient large-fragment knock-in generation in two-cell mouse embryos, highlighting an accelerated platform for complex animal modeling.

Key Points

To establish a standardized, highly efficient protocol for generating large-fragment knock-in mouse models by delivering CRISPR gene-editing components into two-cell-stage embryos.
Microinjected CRISPR gene-editing reagents into mouse embryos at the two-cell stage to leverage high homologous recombination activity during their extended G2 phase.
Employed a Cas9 fusion with monomeric streptavidin (Cas-mSA) paired with a biotinylated PCR donor template (BioPCR) to recruit repair templates directly to targeted double-strand breaks.
Provided systematic protocols covering guide RNA and repair template design, reagent preparation, and two-cell-stage embryonic microinjection.
Delivering editing machinery at the two-cell stage yielded baseline knock-in efficiencies between 10% and 50% for large DNA fragments.
Integrating the Cas9-mSA and biotinylated repair template localization system increased targeting efficiency up to 95%.
Shortened the production timeline for founder knock-in mice to two months.

Cite This Study

Gu et al. (2020) studied this question.

synapsesocial.com/papers/6a170b20b13aec50ea6bcc01 https://doi.org/10.1002/cpmo.67

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January 8, 2020Current Protocols in Mouse Biology

Efficient Generation of Large‐Fragment Knock‐In Mouse Models Using 2‐Cell (2C)‐Homologous Recombination (HR)‐CRISPR

View Full Paper

Ask AI

Bookmark

Authors

BGBin GuSoochow University EPEszter PósfaiPrinceton University MGMarina GertsensteinToronto Centre for Phenogenomics

Discussion

Member takes

Overview

Experimental study demonstrates efficient large-fragment knock-in generation in two-cell mouse embryos, highlighting an accelerated platform for complex animal modeling.

Key Points

To establish a standardized, highly efficient protocol for generating large-fragment knock-in mouse models by delivering CRISPR gene-editing components into two-cell-stage embryos.
Microinjected CRISPR gene-editing reagents into mouse embryos at the two-cell stage to leverage high homologous recombination activity during their extended G2 phase.
Employed a Cas9 fusion with monomeric streptavidin (Cas-mSA) paired with a biotinylated PCR donor template (BioPCR) to recruit repair templates directly to targeted double-strand breaks.
Provided systematic protocols covering guide RNA and repair template design, reagent preparation, and two-cell-stage embryonic microinjection.
Delivering editing machinery at the two-cell stage yielded baseline knock-in efficiencies between 10% and 50% for large DNA fragments.
Integrating the Cas9-mSA and biotinylated repair template localization system increased targeting efficiency up to 95%.
Shortened the production timeline for founder knock-in mice to two months.

Cite This Study

Gu et al. (2020) studied this question.

synapsesocial.com/papers/6a170b20b13aec50ea6bcc01 https://doi.org/10.1002/cpmo.67

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