Reliable CRISPR/Cas9 Genome Engineering in Caenorhabditis elegans Using a Single Efficient sgRNA and an Easily Recognizable Phenotype.
Reliable CRISPR/Cas9 Genome Engineering in Caenorhabditis elegans Using a Single Efficient sgRNA and an Easily Recognizable Phenotype.
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DOI：
10.1534/g3.117.040824
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发表时间：
2017-05-05
期刊：
影响因子：
--
通讯作者：
Boulin T
中科院分区：
文献类型：
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作者：
El Mouridi S;Lecroisey C;Tardy P;Mercier M;Leclercq-Blondel A;Zariohi N;Boulin T
CRISPR/Cas9基因组工程策略允许对秀丽隐杆线虫基因组进行定向修饰，以引入点突变，产生敲除突变体，并插入表位或荧光标签的编码序列。然而，有三个实际方面使这种实验复杂化。首先，单向导RNA（sgRNA）的效率和特异性不能可靠地预测。第二，在缺乏清晰可见或可选择的表型的情况下，检测携带基因组编辑的动物可能具有挑战性。第三，sgRNA靶位点必须在编辑后失活，以避免进一步的双链断裂事件。我们在这里描述了一种策略，通过将高效sgRNA的原型间隔区移植到感兴趣的基因中以使其适合基因组工程来解决这些并发症。这种靶向dpy-10基因的sgRNA以相对高的频率产生基因组编辑。我们证明，移植的protospacer裂解的同时，dpy-10基因。我们的策略产生了无瘢痕的基因组编辑，因为它不再需要在内源性sgRNA靶位点中引入突变。在F1代中可以很容易地鉴定出修饰的后代，这大大减少了通过PCR或表型分析进行测试的动物数量。使用这种策略，我们可靠地产生了精确的缺失突变体，转录报告，并与表位标签和荧光报告基因的翻译融合。特别是，我们在这里报告的第一次使用新的红色荧光蛋白mScarlet在多细胞生物。wrmScarlet，a C. elegans优化的版本，通过在直接比较中显示荧光增加八倍而大大超过了TagRFP-T。
CRISPR/Cas9 genome engineering strategies allow the directed modification of the Caenorhabditis elegans genome to introduce point mutations, generate knock-out mutants, and insert coding sequences for epitope or fluorescent tags. Three practical aspects, however, complicate such experiments. First, the efficiency and specificity of single-guide RNAs (sgRNA) cannot be reliably predicted. Second, the detection of animals carrying genome edits can be challenging in the absence of clearly visible or selectable phenotypes. Third, the sgRNA target site must be inactivated after editing to avoid further double-strand break events. We describe here a strategy that addresses these complications by transplanting the protospacer of a highly efficient sgRNA into a gene of interest to render it amenable to genome engineering. This sgRNA targeting the dpy-10 gene generates genome edits at comparatively high frequency. We demonstrate that the transplanted protospacer is cleaved at the same time as the dpy-10 gene. Our strategy generates scarless genome edits because it no longer requires the introduction of mutations in endogenous sgRNA target sites. Modified progeny can be easily identified in the F1 generation, which drastically reduces the number of animals to be tested by PCR or phenotypic analysis. Using this strategy, we reliably generated precise deletion mutants, transcriptional reporters, and translational fusions with epitope tags and fluorescent reporter genes. In particular, we report here the first use of the new red fluorescent protein mScarlet in a multicellular organism. wrmScarlet, a C. elegans-optimized version, dramatically surpassed TagRFP-T by showing an eightfold increase in fluorescence in a direct comparison.