Generating Zebrafish RNA-Less Mutant Alleles by Deleting Gene Promoters with CRISPR/Cas9.
Generating Zebrafish RNA-Less Mutant Alleles by Deleting Gene Promoters with CRISPR/Cas9.
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DOI：
10.1007/978-1-0716-1847-9_8
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发表时间：
2022
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斑马鱼（Danio rerio）传统上用于正向遗传筛选，在过去十年中，随着TALENS，锌指核酸酶和CRISPR/Cas9的引入，它已成为反向遗传研究的流行模型。出乎意料的是，由这些工具产生的纯合移码突变经常导致比注射靶向相同基因的反义吗啉代寡核苷酸的胚胎中所见的表型更少的外显。对这种差异的一种解释是，一些移码突变导致基因转录物的无义介导的衰变，这是一个可以诱导同源基因表达的过程。这种形式的遗传补偿，称为转录适应，当突变等位基因导致靶基因不产生RNA转录本时，不会发生。这样的无RNA突变体可以通过使用一对指导RNA和Cas9蛋白缺失基因的启动子来产生。在这里，我们提出了一个协议，并使用它来产生等位基因arhgap 29 b和slc 41 a1，缺乏可检测的合子转录。在arhgap 29 b突变体的情况下，新出现的表型不与启动子缺失突变分离，突出了使用这些工具进行脱靶诱变的潜力。总之，本章描述了一种方法，以产生斑马鱼突变体，避免发生在许多移码突变体的遗传补偿的形式。
Danio rerio (zebrafish), traditionally used in forward genetic screens, has in the last decade become a popular model for reverse genetic studies with the introduction of TALENS, zinc finger nucleases, and CRISPR/Cas9. Unexpectedly, homozygous frameshift mutations generated by these tools frequently result in phenotypes that are less penetrant than those seen in embryos injected with antisense morpholino oligonucleotides targeting the same gene. One explanation for the difference is that some frameshift mutations result in nonsense-mediated decay of the gene transcript, a process which can induce expression of homologous genes. This form of genetic compensation, called transcriptional adaptation, does not occur when the mutant allele results in no RNA transcripts being produced from the targeted gene. Such RNA-less mutants can be generated by deleting a gene’s promoter using a pair of guide RNAs and Cas9 protein. Here, we present a protocol and use it to generate alleles of arhgap29b and slc41a1 that lack detectable zygotic transcription. In the case of the arhgap29b mutant, an emerging phenotype did not segregate with the promoter deletion mutation, highlighting the potential for off-target mutagenesis with these tools. In summary, this chapter describes a method to generate zebrafish mutants that avoid a form of genetic compensation that occurs in many frameshift mutants.