Details

Autor(en) / Beteiligte

• Lin, Ying‐Cing
• Pecetta, Simone
• Steichen, Jon M
• Kratochvil, Sven
• Melzi, Eleonora
• Arnold, Johan
• Dougan, Stephanie K
• Wu, Lin
• Kirsch, Kathrin H
• Nair, Usha
• Schief, William R
• Batista, Facundo D

Titel

One‐step CRISPR/Cas9 method for the rapid generation of human antibody heavy chain knock‐in mice

Ist Teil von

• The EMBO journal, 2018-09, Vol.37 (18), p.n/a

Ort / Verlag

England: Blackwell Publishing Ltd

Erscheinungsjahr

2018

Quelle

Alma/SFX Local Collection

Beschreibungen/Notizen

• Here, we describe a one‐step, in vivo CRISPR/Cas9 nuclease‐mediated strategy to generate knock‐in mice. We produced knock‐in (KI) mice wherein a 1.9‐kb DNA fragment bearing a pre‐arranged human B‐cell receptor heavy chain was recombined into the native murine immunoglobulin locus. Our methodology relies on Cas9 nuclease‐induced double‐stranded breaks directed by two sgRNAs to occur within the specific target locus of fertilized oocytes. These double‐stranded breaks are subsequently repaired via homology‐directed repair by a plasmid‐borne template containing the pre‐arranged human immunoglobulin heavy chain. To validate our knock‐in mouse model, we examined the expression of the KI immunoglobulin heavy chains by following B‐cell development and performing single B‐cell receptor sequencing. We optimized this strategy to generate immunoglobulin KI mice in a short amount of time with a high frequency of homologous recombination (30–50%). In the future, we envision that such knock‐in mice will provide much needed vaccination models to evaluate immunoresponses against immunogens specific for various infectious diseases. Synopsis We describe an in vivo, CRISPR/Cas9 nuclease‐mediated strategy to generate knock‐in (KI) mice. A large 1.9‐kb DNA fragment bearing a pre‐arranged human B‐cell receptor heavy chain was recombined into the native murine immunoglobulin locus. We optimized this strategy to generate two independent heavy chain KI mouse models in about 3 weeks with a high frequency of homologous recombination (30–50%). Via a CRISPR/Cas9‐nuclease‐mediated strategy, a 1.9‐kb DNA fragment bearing a pre‐arranged human B‐cell receptor heavy chain was recombined into the native murine immunoglobulin locus. The frequency of recombination is 30–50%. This strategy enabled us to generate knock‐in mice in about 3 weeks. Direct recombination of pre‐arranged human B‐cell receptor into the native immunoglobulin locus in fertilized mouse oocytes provides a facile strategy for obtaining vaccination models for infectious disease.