Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing
Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing
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通过 CRISPR/Cas 介导的基因组编辑将精确的基因修饰引入人类 3PN 胚胎
DOI：
10.1007/s10815-016-0710-8
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发表时间：
2016-05-01
影响因子：
3.1
通讯作者：
Fan, Yong
中科院分区：
文献类型：
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作者：
Kang, Xiangjin;He, Wenyin;Fan, Yong
作为一项强大的基因组工程技术，CRISPR/Cas系统已成功应用于多种物种的基因组修饰。本研究的目的是评估技术并建立在早期人类胚胎中引入精确基因修饰的原则。3PN受精卵注射Cas9信使RNA (mRNA) （100 ng/ μ l）和引导RNA (gRNA) （50 ng/ μ l）。对于oligo注射，将供体oligo-1 （99 bp）或oligo-2 (99 bp) （100 ng/mu l）或dsDonor （1 kb）与Cas9 mRNA （100 ng/mu l）和gRNA （50 ng/mu l）混合注射到胚胎中。通过共注射Cas9 mRNA、grna和供体DNA，我们成功地将自然存在的CCR5 Delta 32等位基因引入早期人类3PN胚胎。然而，在含有工程CCR5 Delta 32等位基因的胚胎中，同一位点的其他等位基因无法完全控制，因为它们要么保持野生型，要么包含indel突变。这项工作对遗传疾病的治疗方法的发展有影响，它表明重大的技术问题仍有待解决。我们主张在全球研究界和伦理界进行严格、彻底的评估和讨论之前，禁止对人类生殖系进行任何基因组编辑应用。
As a powerful technology for genome engineering, the CRISPR/Cas system has been successfully applied to modify the genomes of various species. The purpose of this study was to evaluate the technology and establish principles for the introduction of precise genetic modifications in early human embryos.3PN zygotes were injected with Cas9 messenger RNA (mRNA) (100 ng/mu l) and guide RNA (gRNA) (50 ng/mu l). For oligo-injections, donor oligo-1 (99 bp) or oligo-2 (99 bp) (100 ng/mu l) or dsDonor (1 kb) was mixed with Cas9 mRNA (100 ng/mu l) and gRNA (50 ng/mu l) and injected into the embryos.By co-injecting Cas9 mRNA, gRNAs, and donor DNA, we successfully introduced the naturally occurring CCR5 Delta 32 allele into early human 3PN embryos. In the embryos containing the engineered CCR5 Delta 32 allele, however, the other alleles at the same locus could not be fully controlled because they either remained wild type or contained indel mutations.This work has implications for the development of therapeutic treatments of genetic disorders, and it demonstrates that significant technical issues remain to be addressed. We advocate preventing any application of genome editing on the human germline until after a rigorous and thorough evaluation and discussion are undertaken by the global research and ethics communities.