Greiner Vera, Bou Puerto Regina, Liu Suying, Herbel Christoph, Carmona Ellese M, Goldberg Michael S
Department of Cancer Immunology & Virology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Department of Microbiology & Immunobiology, Harvard Medical School, Boston, MA 02215, USA.
Department of Cancer Immunology & Virology, Dana-Farber Cancer Institute, Boston, MA 02215, USA.
iScience. 2019 Feb 22;12:369-378. doi: 10.1016/j.isci.2019.01.032. Epub 2019 Feb 1.
Vaccination approaches have generally focused on the antigen rather than the resultant antibodies generated, which differ greatly in quality and function between individuals. The ability to replace the variable regions of the native B cell receptor (BCR) heavy and light chain loci with defined recombined sequences of a preferred monoclonal antibody could enable curative adoptive cell transfer. We report CRISPR-mediated homologous recombination (HR) into the BCR of primary human B cells. Ribonucleoprotein delivery enabled editing at the model CXCR4 locus, as demonstrated by T7E1 assay, flow cytometry, and TIDE analysis. Insertion via HR was confirmed by sequencing, cross-boundary PCR, and restriction digest. Optimized conditions were used to achieve HR at the BCR variable heavy and light chains. Insertion was confirmed at the DNA level, and transgene expression from the native BCR promoters was observed. Reprogramming the specificity of antibodies in the genomes of B cells could have clinical importance.
疫苗接种方法通常侧重于抗原而非所产生的抗体，个体之间抗体的质量和功能差异很大。用优选单克隆抗体的特定重组序列替换天然B细胞受体（BCR）重链和轻链基因座的可变区的能力，可能实现治愈性过继性细胞转移。我们报告了CRISPR介导的同源重组（HR）进入原代人B细胞的BCR。核糖核蛋白递送能够在模型CXCR4基因座进行编辑，T7E1分析、流式细胞术和TIDE分析证明了这一点。通过测序、跨界PCR和限制性酶切确认了通过HR的插入。使用优化条件在BCR可变重链和轻链上实现HR。在DNA水平确认了插入，并观察到来自天然BCR启动子的转基因表达。对B细胞基因组中的抗体特异性进行重新编程可能具有临床重要性。
