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

• DOI: 10.15252/embj.201899243
• Publication Date: 2018-08-07T09:55:11Z
• Authors: Ying‐Cing Lin; Simone Pecetta; Jon M Steichen; Sven Kratochvil; Eleonora Melzi; Johan Arnold; Stephanie K Dougan; Lin Wu; Kathrin H Kirsch; Usha Nair; William R Schief; Facundo D Batista
• Keywords: Resource; 0301 basic medicine; B-Lymphocytes; 0303 health sciences; Mice, Transgenic; 16. Peace & justice; 3. Good health; Mice; 03 medical and health sciences; Animals; Humans; Gene Knock-In Techniques; CRISPR-Cas Systems; Immunoglobulin Heavy Chains

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.