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.

在此，我们描述了一种一步法、体内CRISPR/Cas9核酸酶介导的策略来生成基因敲入小鼠。我们培育出了基因敲入（KI）小鼠，其中一个携带预先排列的人B细胞受体重链的1.9kb DNA片段被重组到小鼠天然免疫球蛋白基因座中。我们的方法依赖于Cas9核酸酶诱导的由两个sgRNA引导的双链断裂，使其在受精卵的特定靶基因座内发生。这些双链断裂随后通过含有预先排列的人免疫球蛋白重链的质粒携带模板进行同源定向修复。为了验证我们的基因敲入小鼠模型，我们通过追踪B细胞发育以及进行单个B细胞受体测序来检测基因敲入免疫球蛋白重链的表达。我们对该策略进行了优化，以便在短时间内以较高的同源重组频率（30% - 50%）生成免疫球蛋白基因敲入小鼠。未来，我们设想这类基因敲入小鼠将提供急需的疫苗接种模型，以评估针对各种传染病特异性免疫原的免疫反应。