A rapid and effective method for screening, sequencing and reporter verification of engineered frameshift mutations in zebrafish.
A rapid and effective method for screening, sequencing and reporter verification of engineered frameshift mutations in zebrafish.
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DOI：
10.1242/dmm.026765
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发表时间：
2017-06-01
影响因子：
4.3
通讯作者：
Berman JN
中科院分区：
文献类型：
--
作者：
Prykhozhij SV;Steele SL;Razaghi B;Berman JN
基于簇状规则间隔回文重复序列(CRISPR)/CAS的针对细菌中病原体的适应性免疫已被用于基因组编辑，并应用于斑马鱼(Danio Rerio)以产生蛋白质编码基因的移码突变。虽然已经有方法来检测、量化和测序CRISPR/Cas9诱导的突变，但识别F1杂合子FISH中的突变仍然具有挑战性。此外，对突变进行测序并假设它导致移码并不能证明因果关系，因为可能存在替代的翻译起始点和突变对剪接的潜在影响。斑马鱼蛋白的抗体相对较少，限制了在蛋白质水平上的验证，这加剧了这个问题。为了解决这些问题，我们制定了一项详细的方案来筛选F1突变携带者，并克隆和测序鉴定突变。为了验证突变是否真的会导致移码，我们建立了一个荧光报告系统，可以基于野生型和突变型cDNA片段的克隆及其表达水平来检测移码效率。作为原理的证明，我们将这一策略应用于CRISPR/Cas9诱导的三个pycr1a、CHD7和hace1基因突变。在pycr1a中插入7个核苷酸导致了CRISPR/Cas9诱导的斑马鱼突变中第一次观察到外显子跳跃。然而，在这三个突变基因中，荧光报告显示只有在CHD7的两个核苷酸缺失的情况下才有有效的移码，这表明在另外两个突变中存在替代翻译位点的活性，尽管pycr1a外显子跳过缺失可能是有害的。本文提供了一种鉴定斑马鱼移码突变的方法，并强调了在抗体检测不到蛋白质丢失的情况下，在mRNA水平上检查突变并利用荧光记者验证其对翻译的影响的重要性。摘要：一种基于CRISPR/Cas9的斑马鱼荧光报告技术的新方法，以证实该模型中的基因功能缺失。
Clustered regularly interspaced palindromic repeats (CRISPR)/Cas-based adaptive immunity against pathogens in bacteria has been adapted for genome editing and applied in zebrafish (Danio rerio) to generate frameshift mutations in protein-coding genes. Although there are methods to detect, quantify and sequence CRISPR/Cas9-induced mutations, identifying mutations in F1 heterozygous fish remains challenging. Additionally, sequencing a mutation and assuming that it causes a frameshift does not prove causality because of possible alternative translation start sites and potential effects of mutations on splicing. This problem is compounded by the relatively few antibodies available for zebrafish proteins, limiting validation at the protein level. To address these issues, we developed a detailed protocol to screen F1 mutation carriers, and clone and sequence identified mutations. In order to verify that mutations actually cause frameshifts, we created a fluorescent reporter system that can detect frameshift efficiency based on the cloning of wild-type and mutant cDNA fragments and their expression levels. As proof of principle, we applied this strategy to three CRISPR/Cas9-induced mutations in pycr1a, chd7 and hace1 genes. An insertion of seven nucleotides in pycr1a resulted in the first reported observation of exon skipping by CRISPR/Cas9-induced mutations in zebrafish. However, of these three mutant genes, the fluorescent reporter revealed effective frameshifting exclusively in the case of a two-nucleotide deletion in chd7, suggesting activity of alternative translation sites in the other two mutants even though pycr1a exon-skipping deletion is likely to be deleterious. This article provides a protocol for characterizing frameshift mutations in zebrafish, and highlights the importance of checking mutations at the mRNA level and verifying their effects on translation by fluorescent reporters when antibody detection of protein loss is not possible. Summary: A new CRISPR/Cas9-based method of fluorescent reporter technology in zebrafish to confirm loss of gene function in this model.