Cystinuria Type A is a relatively common genetic kidney disease occurring in 1 in 7,000 people worldwide that results from mutation of the cystine transporter rBAT encoded by Slc3a1. We used CRISPR/Cas9 technology to engineer cystinuria Type A mice via genome editing of the C57BL/6NHsd background. These mice are an improvement on currently available models as they are on a coisogenic genetic background and have a single defined mutation. In order to use albinism to track Cas9 activity, we co‐injected gRNAs targeting Slc3a1 and tyrosinase (Tyr) with Cas9 expressing plasmid DNA into mouse embryos. Two different Slc3a1 mutational alleles were derived, with homozygous mice of both demonstrating elevated urinary cystine levels, cystine crystals, and bladder stones. We used whole genome sequencing to evaluate for potential off‐target editing. No off‐target indels were observed for the top 10 predicted off‐targets for Slc3a1 or Tyr. Therefore, we used CRISPR/Cas9 to generate coisogenic albino cystinuria Type A mice that could be used for in vivo imaging, further study, or developing new treatments of cystinuria.

中文翻译：

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白化胱氨酸尿症 A 型小鼠的 CRISPR/Cas9 工程。

A 型胱氨酸尿症是一种相对常见的遗传性肾脏疾病，全世界每 7,000 人中就有 1 人发生这种疾病，这是由Slc3a1编码的胱氨酸转运蛋白 rBAT 突变引起的。我们使用 CRISPR/Cas9 技术通过对 C57BL/6NHsd 背景的基因组编辑来改造 A 型胱氨酸尿症小鼠。这些小鼠是对当前可用模型的改进，因为它们处于同源遗传背景并具有单一定义的突变。为了利用白化病追踪 Cas9 的活性，我们将靶向Slc3a1和酪氨酸酶 ( Tyr ) 的 gRNA 与表达 Cas9 的质粒 DNA 共同注射到小鼠胚胎中。两种不同的 Slc3a1突变等位基因被衍生，纯合子小鼠均表现出尿胱氨酸水平升高、胱氨酸晶体和膀胱结石。我们使用全基因组测序来评估潜在的脱靶编辑。对于Slc3a1或Tyr的前 10 个预测脱靶，没有观察到脱靶插入缺失。因此，我们使用 CRISPR/Cas9 生成了 A 型同源性白化型胱氨酸尿症小鼠，可用于体内成像、进一步研究或开发胱氨酸尿症的新疗法。