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A high-efficiency and versatile CRISPR/Cas9-mediated HDR-based biallelic editing system

[基于同源介导修复 (HDR) 的 CRISPR/Cas9 双等位编辑系统的构建]

作     者:Xinyi LI Bing SUN Hongrun QIAN Jinrong MA Magdalena PAOLINO Zhiying ZHANG Xinyi LI;Bing SUN;Hongrun QIAN;Jinrong MA;Magdalena PAOLINO;Zhiying ZHANG

作者机构:College of Animal Science and TechnologyNorthwest A&F UniversityYangling 712100China Karolinska InstituteDepartment of Medicine SolnaCenter for Molecular MedicineKarolinska University Hospital Solna17176 StockholmSweden 

出 版 物:《Journal of Zhejiang University-Science B(Biomedicine & Biotechnology)》 (浙江大学学报(英文版)B辑(生物医学与生物技术))

年 卷 期:2022年第23卷第2期

页      面:141-152页

核心收录:

学科分类:0710[理学-生物学] 07[理学] 08[工学] 09[农学] 071007[理学-遗传学] 0901[农学-作物学] 0836[工学-生物工程] 090102[农学-作物遗传育种] 

基  金:supported by the National Science and Technology Major Project of China(No.2018ZX08010-09B) the Swedish Research Council(No.NE 2016-04458) the Ragnar S?derberg Foundation(No.M21/17),Sweden 

主  题:Biallelic editing CRISPR/Cas9 Homology-directed repair(HDR) Homozygote 

摘      要:Clustered regulatory interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9 nuclease(Cas9),the third-generation genome editing tool,has been favored because of its high efficiency and clear system *** this technology,the introduced double-strand breaks(DSBs)are mainly repaired by non-homologous end joining(NHEJ)or homology-directed repair(HDR)*** high-fidelity HDR pathway is used for genome modification,which can introduce artificially controllable insertions,deletions,or substitutions carried by the donor *** high-level knock-out can be easily achieved by NHEJ,accurate HDR-mediated knock-in remains a technical *** most circumstances,although both alleles are broken by endonucleases,only one can be repaired by HDR,and the other one is usually recombined by *** gene function studies or disease model establishment,biallelic editing to generate homozygous cell lines and homozygotes is needed to ensure consistent ***,there is an urgent need for an efficient biallelic editing ***,we developed three pairs of integrated selection systems,where each of the two selection cassettes contained one drug-screening gene and one fluorescent *** by homologous arms containing the mutated sequences,the selection cassettes were integrated into the target site,mediated by CRISPR/Cas9-induced *** targeted cell clones were massively enriched by fluorescent microscopy after screening for drug *** tested this novel method on the amyloid precursor protein(APP)and presenilin 1(PSEN1)loci and demonstrated up to 82.0%biallelic editing efficiency after *** results indicate that this strategy can provide a new efficient approach for biallelic editing and lay a foundation for establishment of an easier and more efficient disease model.

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