One-step CRISPR/Cas9 method for the rapid generation of human antibody heavy chain knock-in mice
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.
Source: EMBO Journal - Category: Molecular Biology Authors: Lin, Y.-C., Pecetta, S., Steichen, J. M., Kratochvil, S., Melzi, E., Arnold, J., Dougan, S. K., Wu, L., Kirsch, K. H., Nair, U., Schief, W. R., Batista, F. D. Tags: Genetics, Gene Therapy & Genetic Disease, Immunology Resource Source Type: research
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