Targeted disruption of the endogenous zebrafish rhodopsin locus as models of rapid rod photoreceptor degeneration
Targeted disruption of the endogenous zebrafish rhodopsin locus as models of rapid rod photoreceptor degeneration
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发表时间：
2018-08
期刊：
影响因子：
2.2
通讯作者：
C. Zelinka;Mailin Sotolongo-Lopez;J. M. Fadool
中科院分区：
文献类型：
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作者：
C. Zelinka;Mailin Sotolongo-Lopez;J. M. Fadool
目的视网膜色素变性（Retinitispigmentosa，RP）是一组遗传性疾病，导致感光细胞变性，导致失明。RP与超过70个可能显示显性或隐性遗传模式的基因座相关，但编码视色素视紫红质（RHO）的基因突变是最常见的原因。为了在斑马鱼中开发精确的突变作为感光细胞退化的新模型，我们描述了一系列新的成簇规则间隔短回文重复序列（CRISPR）/Cas9诱导的插入和缺失（indel）突变在主要斑马鱼rho基因座rh 1 -1中的产生和种系传递。方法将体外转录的编码Cas9的mRNA和一种单一的指导RNA（gRNA）显微注射到单细胞或双细胞阶段的斑马鱼胚胎中。通过限制性片段长度多态性（RFLP）和DNA序列分析检测注射胚胎和后代的突变。用视杆细胞特异性抗体和视锥细胞特异性抗体进行免疫标记，以检测组织学和细胞变化。结果使用靶向rh 1 -1高度保守区域的gRNA，恢复了一系列导致视杆细胞快速变性的显性和隐性等位基因。未观察到对视锥细胞的影响。靶向rh 1 -1的5 '编码序列导致了几个与疾病相关等位基因相似的插入缺失的恢复。导致过早终止密码子（T17*）的移码突变在纯合性时导致视杆变性。免疫印迹和荧光标记的Rho特异性抗体表明，这确实是一个无效等位基因，说明Rho的表达是必不可少的杆生存。回收了两个框内突变，其破坏了N15处高度保守的N-连接糖基化共有序列。任何一个等位基因的杂合子表现出快速杆变性。靶向rh 1 -1的3′-编码区导致恢复了编码保守VSPA分选序列上游提前终止密码子（S347*）的等位基因和破坏S339处推定磷酸化位点的第二个符合读码框等位基因。这两个等位基因导致杆死亡的显性遗传模式。靶向序列丢失后，Rho的免疫标记不再局限于视杆外节，也定位于质膜。结论CRISPR/Cas9的基因打靶效率，加上RP相关的大量突变，为在斑马鱼中快速分离新等位基因提供了背景。这些新的细胞系将提供急需的体内模型，用于高通量筛选保护光感受器不变性的化合物或基因。
Purpose Retinitis pigmentosa (RP) is a collection of genetic disorders that results in the degeneration of light-sensitive photoreceptor cells, leading to blindness. RP is associated with more than 70 loci that may display dominant or recessive modes of inheritance, but mutations in the gene encoding the visual pigment rhodopsin (RHO) are the most frequent cause. In an effort to develop precise mutations in zebrafish as novel models of photoreceptor degeneration, we describe the generation and germline transmission of a series of novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-induced insertion and deletion (indel) mutations in the major zebrafish rho locus, rh1–1. Methods One- or two-cell staged zebrafish embryos were microinjected with in vitro transcribed mRNA encoding Cas9 and a single guide RNA (gRNA). Mutations were detected by restriction fragment length polymorphism (RFLP) and DNA sequence analyses in injected embryos and offspring. Immunolabeling with rod- and cone-specific antibodies was used to test for histological and cellular changes. Results Using gRNAs that targeted highly conserved regions of rh1–1, a series of dominant and recessive alleles were recovered that resulted in the rapid degeneration of rod photoreceptors. No effect on cones was observed. Targeting the 5′-coding sequence of rh1–1 led to the recovery of several indels similar to disease-associated alleles. A frame shift mutation leading to a premature stop codon (T17*) resulted in rod degeneration when brought to homozygosity. Immunoblot and fluorescence labeling with a Rho-specific antibody suggest that this is indeed a null allele, illustrating that the Rho expression is essential for rod survival. Two in-frame mutations were recovered that disrupted the highly conserved N-linked glycosylation consensus sequence at N15. Larvae heterozygous for either of the alleles demonstrated rapid rod degeneration. Targeting of the 3′-coding region of rh1–1 resulted in the recovery of an allele encoding a premature stop codon (S347*) upstream of the conserved VSPA sorting sequence and a second in-frame allele that disrupted the putative phosphorylation site at S339. Both alleles resulted in rod death in a dominant inheritance pattern. Following the loss of the targeting sequence, immunolabeling for Rho was no longer restricted to the rod outer segment, but it was also localized to the plasma membrane. Conclusions The efficiency of CRISPR/Cas9 for gene targeting, coupled with the large number of mutations associated with RP, provided a backdrop for the rapid isolation of novel alleles in zebrafish that phenocopy disease. These novel lines will provide much needed in-vivo models for high throughput screens of compounds or genes that protect from photoreceptor degeneration.