کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
6490148 | 1416963 | 2018 | 28 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Speed genome editing by transient CRISPR/Cas9 targeting and large DNA fragment deletion
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کلمات کلیدی
FACSeGFPCAEECFPsingle-guide RNASingle cell sortingCRISPR/Cas9 - CRISPR / Cas9sgRNA - scRNAT2A - T2enhanced cyan fluorescent protein - افزایش پروتئین فلورسنت سیانوژنCapillary array electrophoresis - الکتروفورز آرایه مویرگیFluorescent reporter - خبرنگار فلورسنتfluorescence-activated cell sorting - دسته بندی سلول های فعال فلورسنسenhanced green fluorescent protein - پروتئین فلورسنت سبز افزایش یافته است
موضوعات مرتبط
مهندسی و علوم پایه
مهندسی شیمی
بیو مهندسی (مهندسی زیستی)
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
Genetic engineering of cell lines and model organisms has been facilitated enormously by the CRISPR/Cas9 system. However, in cell lines it remains labor intensive and time consuming to obtain desirable mutant clones due to the difficulties in isolating the mutated clones and sophisticated genotyping. In this study, we have validated fluorescent protein reporter aided cell sorting which enables the isolation of maximal diversity in mutant cells. We further applied two spectrally distinct fluorescent proteins DsRed2 and ECFP as reporters for independent CRISPR/Cas9 mediated targeting, which allows for one-cell-one-well sorting of the mutant cells. Because of ultra-high efficiency of the CRISPR/Cas9 system with dual reporters and large DNA fragment deletion resulting from independent loci cleavage, monoclonal mutant cells could be easily identified by conventional PCR. In the speed genome editing method presented here, sophisticated genotyping methods are not necessary to identify loss of function mutations after CRISPR/Cas9 genome editing, and desirable loss of function mutant clones could be obtained in less than one month following transfection.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Biotechnology - Volume 281, 10 September 2018, Pages 11-20
Journal: Journal of Biotechnology - Volume 281, 10 September 2018, Pages 11-20
نویسندگان
Jing Luo, Liaoxun Lu, Yanrong Gu, Rong Huang, Lin Gui, Saichao Li, Xinhui Qi, Wenping Zheng, Tianzhu Chao, Qianqian Zheng, Yinming Liang, Lichen Zhang,