CRISPR/Cas9: An inexpensive, efficient loss of function tool to screen human disease genes in Xenopus.
CRISPR/Cas9: An inexpensive, efficient loss of function tool to screen human disease genes in Xenopus.
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DOI：
10.1016/j.ydbio.2015.11.003
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发表时间：
2015-12-15
影响因子：
2.7
通讯作者：
Khokha MK
中科院分区：
文献类型：
--
作者：
Bhattacharya D;Marfo CA;Li D;Lane M;Khokha MK
先天性畸形是美国和欧洲婴儿死亡的主要原因。由于人类基因组学的快速发展，我们现在可以有效地识别可能导致这些患者疾病的序列变异。然而，确定疾病的因果关系仍然是一个挑战。此外，在先天性心脏病的情况下，许多已鉴定的候选基因要么是胚胎发育的新基因，要么没有已知的功能。因此，迫切需要开发廉价和有效的技术来筛选这些候选基因的疾病表型在模型系统中，并进行功能研究，以揭示其在发展中的作用。为此，我们试图测试基于F0 CRISPR的基因编辑作为青蛙模式生物热带爪蟾（Xenopus tropicalis）中疾病表型复制的功能丧失策略。我们证明，CRISPR/ Cas9系统可以在受精后几小时内有效地修饰F0代中的两个等位基因，甚至在几乎所有测试的情况下，重演与吗啉代寡核苷酸（MO）敲除高度相似的早期疾病表型。我们发现注射Cas9蛋白比cas9 mRNA显著更有效且毒性更低。我们的结论是，基于CRISPR的F0基因修饰在X. Tropicalis是有效的和成本有效的，并且容易概括疾病和MO表型。
Congenital malformations are the major cause of infant mortality in the US and Europe. Due to rapid advances in human genomics, we can now efficiently identify sequence variants that may cause disease in these patients. However, establishing disease causality remains a challenge. Additionally, in the case of congenital heart disease, many of the identified candidate genes are either novel to embryonic development or have no known function. Therefore, there is a pressing need to develop inexpensive and efficient technologies to screen these candidate genes for disease phenocopy in model systems and to perform functional studies to uncover their role in development. For this purpose, we sought to test F0 CRISPR based gene editing as a loss of function strategy for disease phenocopy in the frog model organism, Xenopus tropicalis. We demonstrate that the CRISPR/ Cas9 system can efficiently modify both alleles in the F0 generation within a few hours post fertilization, recapitulating even early disease phenotypes that are highly similar to knockdowns from morpholino oligos (MOs) in nearly all cases tested. We find that injecting Cas9 protein is dramatically more efficacious and less toxic than cas9 mRNA. We conclude that CRISPR based F0 gene modification in X. tropicalis is efficient and cost effective and readily recapitulates disease and MO phenotypes.