Precise gene editing of model organisms is required for accurately modeling human diseases and deciphering gene functions. In this study, we used a pair of guide RNAs (sgRNAs), which in vitro transcribed along with other CRISPR RNA components, to generate two cleavage sites flanking pig GJB2 (pGJB2) CDS. By using long single-stranded DNAs (lssDNA) as homology-directed repair (HDR) templates, we efficiently obtained two gene-edited pigs, of which GJB2 CDS replaced with CDSs containing human GJB2 c.235delC mutation and orthologous human p.V37I mutation, respectively. These mutations were commonly observed in patients with hearing loss. Genetic analysis of the two gene-edited pigs showed that the HDR-derived gene-editing efficiency were as high as 80% (4/5) and 50% (2/4), respectively. While no mutation was observed in the group of single cutting with one sgRNA covering the 235th nucleotide C in pGJB2 CDS, using a short single-stranded oligo DNA containing c.235delC mutation as HDR template. Extra experiments proved that the intended mutations were successfully transmitted to offspring or extensively integrated into various tissues including gonad of founder pigs. Our work indicated that the new “double cutting with lssDNA template” gene editing method can expand sgRNA selection scope and avoids direct cutting of gene CDS. Additionally, can introduce precise mutations into mammalian genomic sites, especially those with unavailable proper protospacer sequence or being resistant to gene editing. Moreover, this method can be performed with CRISPR RNA reagents instead of CRISPR ribonucleoproteins applied in previous reports.

精确地模拟人类疾病和破译基因功能需要对模型生物进行精确的基因编辑。在这项研究中，我们使用了一对引导 RNA (sgRNA)，它们与其他 CRISPR RNA 成分一起在体外转录，以产生两个位于猪GJB2 ( pGJB2 ) CDS侧翼的切割位点。通过使用长单链DNA（lssDNA）作为同源定向修复（HDR）模板，我们有效地获得了两只基因编辑猪，其中GJB2 CDS被含有人GJB2 c.235delC突变和直系同源人p.V37I的CDS取代突变，分别。这些突变通常在听力损失患者中观察到。对两只基因编辑猪的遗传分析表明，HDR衍生的基因编辑效率分别高达80%（4/5）和50%（2/4）。而在用一个sgRNA覆盖pGJB2 CDS中第235个核苷酸C的单切割组中未观察到突变，使用含有c.235delC的短单链寡核苷酸DNA突变作为 HDR 模板。额外的实验证明，预期的突变成功地传递给后代或广泛整合到各种组织中，包括创始猪的性腺。我们的工作表明，新的“lssDNA模板双切割”基因编辑方法可以扩大sgRNA的选择范围，避免直接切割基因CDS。此外，可以将精确的突变引入哺乳动物基因组位点，尤其是那些无法获得正确的原始间隔序列或对基因编辑具有抗性的位点。此外，这种方法可以用 CRISPR RNA 试剂代替以前报告中应用的 CRISPR 核糖核蛋白进行。