In vivo genome editing by NanoMEDIC CRISPR-Cas9/sgRNA delivery into a Luc-reporter mosue model.

We first generated a transgenic luciferase reporter mouse model to monitor human Dystrophin gene exon 45 skipping activity. Two CRISPR-sgRNAs and Cas9 protein were delivered by NanoMEDIC technology into gastrocnemius muscle of the reporter mice, and genomic DNAs were extracted 189 days post-injection. The target region was PCR amplified and subjected to Illumina MiSeq amplicon sequencing (300 bp ×2). Each sample (PBS injection: 3 mice, NanoMEDIC injection: 3 mice) was spitted by unique 4 bp index, and pair-end reads were merged by FLASH2 software.

本研究首先构建了转基因荧光素酶报告小鼠模型，用于监测人类肌营养不良蛋白（Dystrophin）基因第45外显子的剪接跳过活性。将两条CRISPR单引导RNA（CRISPR-sgRNAs）与Cas9蛋白通过NanoMEDIC技术递送至报告小鼠的腓肠肌，并于注射后189天提取基因组DNA。对目标区域进行PCR扩增后，采用Illumina MiSeq扩增子测序（300 bp×2）进行检测。每个样本（PBS注射组：3只小鼠，NanoMEDIC注射组：3只小鼠）通过唯一的4 bp索引进行标签拆分，并使用FLASH2软件合并双端测序读段。