CRISPR/Cas9 engineering of albino cystinuria Type A mice.
CRISPR/Cas9 engineering of albino cystinuria Type A mice.
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DOI：
10.1002/dvg.23357
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发表时间：
2020-05
期刊：
影响因子：
--
通讯作者：
Wilson MH
中科院分区：
文献类型：
--
作者：
Beckermann TM;Welch RC;Williams FM;Mortlock DP;Sha F;Ikizler TA;Woodard LE;Wilson MH
A型胱氨酸尿症是一种相对常见的遗传性肾脏疾病，全球发病率为1 / 7000，由Slc3a1编码的胱氨酸转运体rBAT突变引起。我们使用CRISPR/Cas9技术通过对C57BL/6NHsd背景基因进行基因组编辑来改造A型胱氨酸尿鼠。这些小鼠是对现有模型的改进，因为它们具有异基因遗传背景，并且具有单一定义的突变。为了利用白化病追踪Cas9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。得到了两个不同的Slc3a1突变等位基因，纯合子小鼠均表现出尿胱氨酸水平升高、胱氨酸结晶和膀胱结石。我们使用全基因组测序来评估潜在的脱靶编辑。Slc3a1或Tyr的前10个预测脱靶指标均未观察到脱靶指标。因此，我们利用CRISPR/Cas9技术，生成了结缔组织基因白化胱氨酸尿A型小鼠，可用于体内成像、进一步研究或开发胱氨酸尿的新治疗方法。
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 ten 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.
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