کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
8467783 1549559 2017 12 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Optimization of CRISPR/Cas9 genome editing for loss-of-function in the early chick embryo
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی بیولوژی سلول
پیش نمایش صفحه اول مقاله
Optimization of CRISPR/Cas9 genome editing for loss-of-function in the early chick embryo
چکیده انگلیسی
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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Developmental Biology - Volume 432, Issue 1, 1 December 2017, Pages 86-97
نویسندگان
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