CNRS, INSERM, Université de Strasbourg, CELPHEDIA, PHENOMIN-Institut Clinique de la Souris (ICS), 1 rue Laurent Fries, 67404 Illkirch Graffenstaden, France.
CNRS, INSERM, Université de Strasbourg, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Department of Translational Medicine and Neurogenetics, 1 rue Laurent Fries, 67404 Illkirch Graffenstaden, France.
Genes (Basel). 2023 Feb 3;14(2):401. doi: 10.3390/genes14020401.
The French mouse clinic (Institut Clinique de la Souris; ICS) has produced more than 2000 targeting vectors for 'à la carte' mutagenesis in C57BL/6N mice. Although most of the vectors were used successfully for homologous recombination in murine embryonic stem cells (ESCs), a few have failed to target a specific locus after several attempts. We show here that co-electroporation of a CRISPR plasmid with the same targeting construct as the one that failed previously allows the systematic achievement of positive clones. A careful validation of these clones is, however, necessary as a significant number of clones (but not all) show a concatemerization of the targeting plasmid at the locus. A detailed Southern blot analysis permitted characterization of the nature of these events as standard long-range 5' and 3' PCRs were not able to distinguish between correct and incorrect alleles. We show that a simple and inexpensive PCR performed prior to ESC amplification allows detection and elimination of those clones with concatemers. Finally, although we only tested murine ESCs, our results highlight the risk of mis-validation of any genetically modified cell line (such as established lines, induced pluripotent stem cells or those used for ex vivo gene therapy) that combines the use of CRISPR/Cas9 and a circular double-stranded donor. We strongly advise the CRISPR community to perform a Southern blot with internal probes when using CRISPR to enhance homologous recombination in any cell type, including fertilized oocytes.
法国小鼠临床研究所（Institut Clinique de la Souris；ICS）已经生产了超过 2000 个用于 C57BL/6N 小鼠“点菜式”诱变的靶向载体。尽管大多数载体在小鼠胚胎干细胞（ESCs）中成功用于同源重组，但有少数载体在多次尝试后未能靶向特定基因座。我们在这里展示，与以前失败的靶向构建体相同的 CRISPR 质粒的共电穿孔允许系统地获得阳性克隆。然而，需要仔细验证这些克隆，因为大量克隆（但不是全部）显示在靶基因座处靶向质粒的串联。详细的 Southern blot 分析允许对这些事件的性质进行表征，因为标准的长距离 5'和 3'PCR 无法区分正确和错误的等位基因。我们表明，在 ESC 扩增之前进行的简单且廉价的 PCR 可以检测并消除那些具有串联体的克隆。最后，尽管我们仅测试了小鼠 ESCs，但我们的结果强调了任何使用 CRISPR/Cas9 和环状双链供体的基因修饰细胞系（例如已建立的细胞系、诱导多能干细胞或用于体外基因治疗的细胞系）都存在错误验证的风险。我们强烈建议 CRISPR 社区在使用 CRISPR 增强任何细胞类型（包括受精卵）中的同源重组时，使用内部探针进行 Southern blot。
