Optimization of CRISPR/Cas9 genome editing for loss-of-function in the early chick embryo.
Optimization of CRISPR/Cas9 genome editing for loss-of-function in the early chick embryo.
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DOI：
10.1016/j.ydbio.2017.08.036
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发表时间：
2017-12-01
影响因子：
2.7
通讯作者：
Bronner ME
中科院分区：
文献类型：
--
作者：
Gandhi S;Piacentino ML;Vieceli FM;Bronner ME
CRISPR/CAS9的出现使基因组编辑几乎可以在任何生物体中进行，包括那些以前不能进行基因操作的生物体。在这里，我们提出了CRISPR/CAS9的优化，通过三重策略提高了应用于早期禽类胚胎的效率。首先，我们利用Cas9蛋白两侧的两个核定位信号序列来改进核定位。其次，我们使用了一种改进的引导RNA(GRNA)支架，它避免了转录的提前终止和不稳定的Cas9-gRNA相互作用。第三，我们使用了鸡特异性U6启动子，其gRNA表达比之前使用的人U6高4倍。为了快速筛选体内应用的gRNAs，我们还建立了一个结构性表达Cas9的鸡成纤维细胞系。作为原理的证明，我们在早期鸡胚中进行了基于电穿孔的功能丧失研究，以敲除Pax7和Sox10，这两个转录因子在神经脊发育中具有已知的功能。结果表明，CRISPR/Cas9介导的缺失会导致它们各自的蛋白质和转录本以及预测的下游靶点的丢失。综上所述，这些结果揭示了这种优化的CRISPR/Cas9方法以一种重复性好、健壮和特异的方式在鸡胚中进行靶向基因敲除的实用性。
The advent of CRISPR/Cas9 has made genome editing possible in virtually any organism, including those not previously amenable to genetic manipulations. Here, we present an optimization of CRISPR/Cas9 for application to early avian embryos with improved efficiency via a three-fold strategy. First, we employed Cas9 protein flanked with two nuclear localization signal sequences for improved nuclear localization. Second, we used a modified guide RNA (gRNA) scaffold that obviates premature termination of transcription and unstable Cas9-gRNA interactions. Third, we used a chick-specific U6 promoter that yields 4-fold higher gRNA expression than the previously utilized human U6. For rapid screening of gRNAs for in vivo applications, we also generated a chicken fibroblast cell line that constitutively expresses Cas9. As proof of principle, we performed electroporation-based loss-of-function studies in the early chick embryo to knock out Pax7 and Sox10, key transcription factors with known functions in neural crest development. The results show that CRISPR/Cas9-mediated deletion causes loss of their respective proteins and transcripts, as well as predicted downstream targets. Taken together, the results reveal the utility of this optimized CRISPR/Cas9 method for targeted gene knockout in chicken embryos in a manner that is reproducible, robust and specific.
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影响因子：
2.7
作者：
Ezin, Akouavi M.;Fraser, Scott E.;Bronner-Fraser, Marianne
通讯作者：
Bronner-Fraser, Marianne
影响因子：
7
作者：
Cho SW;Kim S;Kim Y;Kweon J;Kim HS;Bae S;Kim JS
通讯作者：
Kim JS
影响因子：
46.9
作者：
Doench JG;Fusi N;Sullender M;Hegde M;Vaimberg EW;Donovan KF;Smith I;Tothova Z;Wilen C;Orchard R;Virgin HW;Listgarten J;Root DE
通讯作者：
Root DE
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
CRISPR-CAS9介导的基因失活的高度活性SGRNA的合理设计。
影响因子：
46.9
作者：
Doench, John G.;Hartenian, Ella;Graham, Daniel B.;Tothova, Zuzana;Hegde, Mudra;Smith, Ian;Sullender, Meagan;Ebert, Benjamin L.;Xavier, Ramnik J.;Root, David E.
通讯作者：
Root, David E.