An Efficient Genome Editing Strategy To Generate Putative Null Mutants in Caenorhabditis elegans Using CRISPR/Cas9.
An Efficient Genome Editing Strategy To Generate Putative Null Mutants in Caenorhabditis elegans Using CRISPR/Cas9.
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使用CRISPR/CAS9在秀丽隐杆线虫中产生推定的无效突变体的有效基因组编辑策略。
DOI：
10.1534/g3.118.200662
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发表时间：
2018-11-06
期刊：
影响因子：
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通讯作者：
Sternberg PW
中科院分区：
文献类型：
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作者：
Wang H;Park H;Liu J;Sternberg PW
突变体对于分析基因功能是必不可少的。在这里，我们描述了一种简单有效的方法来产生秀丽隐杆线虫无效突变体，使用CRISPR/Cas9和短单链DNA寡核苷酸修复模板将通用的43个核苷酸长的敲入盒（STOP-IN）插入靶基因的早期外显子中。该STOP-IN盒在所有三个阅读框中都具有终止密码子，并导致移码，这将产生推定的无效突变，而不管外显子中插入位置的阅读框如何。STOP-IN盒还含有外源Cas9靶位点，其允许进一步的基因组编辑，并提供了简化通过PCR鉴定成功插入事件的独特序列。作为概念证明，我们在aex-2中的Cas9靶位点处插入STOP-IN盒，以通过注射预组装的Cas9核糖核蛋白和短的合成单链DNA修复模板来产生新的推定无效等位基因，所述短的合成单链DNA修复模板含有STOP-IN盒和与Cas9切割位点侧翼的序列相同的两个约35个核苷酸长的同源臂。我们发现，这些新的aex-2等位基因表型模仿现有的aex-2功能丧失等位基因。我们进一步表明，新的aex-2无效等位基因可以通过靶向STOP-IN盒中包括的外源Cas9切割位点并提供单链野生型DNA修复寡核苷酸而恢复回野生型序列。我们应用我们的STOP-IN方法为另外20个基因产生新的假定无效突变体，包括三个咽肌特异性基因（clik-1，clik-2和clik-3），并报告了基于我们筛选的动物的高插入率（46%）。我们发现，无效突变的clik-2造成隐性致死性与严重的泵送缺陷和clik-3无效突变体有一个温和的泵送缺陷，而clik-1是pumping。我们期望使用STOP-IN盒的敲入方法将有助于产生新的无效突变体，以了解C.线虫和其他遗传模式生物。
Null mutants are essential for analyzing gene function. Here, we describe a simple and efficient method to generate Caenorhabditis elegans null mutants using CRISPR/Cas9 and short single stranded DNA oligo repair templates to insert a universal 43-nucleotide-long knock-in cassette (STOP-IN) into the early exons of target genes. This STOP-IN cassette has stop codons in all three reading frames and leads to frameshifts, which will generate putative null mutations regardless of the reading frame of the insertion position in exons. The STOP-IN cassette also contains an exogenous Cas9 target site that allows further genome editing and provides a unique sequence that simplifies the identification of successful insertion events via PCR. As a proof of concept, we inserted the STOP-IN cassette at a Cas9 target site in aex-2 to generate new putative null alleles by injecting preassembled Cas9 ribonucleoprotein and a short synthetic single stranded DNA repair template containing the STOP-IN cassette and two ∼35-nucleotide-long homology arms identical to the sequences flanking the Cas9 cut site. We showed that these new aex-2 alleles phenocopied an existing loss-of-function allele of aex-2. We further showed that the new aex-2 null alleles could be reverted back to the wild-type sequence by targeting the exogenous Cas9 cut site included in the STOP-IN cassette and providing a single stranded wild-type DNA repair oligo. We applied our STOP-IN method to generate new putative null mutants for 20 additional genes, including three pharyngeal muscle-specific genes (clik-1, clik-2, and clik-3), and reported a high insertion rate (46%) based on the animals we screened. We showed that null mutations of clik-2 cause recessive lethality with a severe pumping defect and clik-3 null mutants have a mild pumping defect, while clik-1 is dispensable for pumping. We expect that the knock-in method using the STOP-IN cassette will facilitate the generation of new null mutants to understand gene function in C. elegans and other genetic model organisms.