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On-line Access: 2022-02-17

Received: 2021-02-27

Revision Accepted: 2021-06-20

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Zhiying ZHANG


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Journal of Zhejiang University SCIENCE B 2022 Vol.23 No.2 P.141-152


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

Author(s):  Xinyi LI, Bing SUN, Hongrun QIAN, Jinrong MA, Magdalena PAOLINO, Zhiying ZHANG

Affiliation(s):  College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China; more

Corresponding email(s):   zhangzhy@nwafu.edu.cn

Key Words:  Biallelic editing, CRISPR/Cas9, Homology-directed repair (HDR), Homozygote

Xinyi LI, Bing SUN, Hongrun QIAN, Jinrong MA, Magdalena PAOLINO, Zhiying ZHANG. A high-efficiency and versatile CRISPR/Cas9-mediated HDR-based biallelic editing system[J]. Journal of Zhejiang University Science B, 2022, 23(2): 141-152.

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author="Xinyi LI, Bing SUN, Hongrun QIAN, Jinrong MA, Magdalena PAOLINO, Zhiying ZHANG",
journal="Journal of Zhejiang University Science B",
publisher="Zhejiang University Press & Springer",

%0 Journal Article
%T A high-efficiency and versatile CRISPR/Cas9-mediated HDR-based biallelic editing system
%A Xinyi LI
%A Bing SUN
%A Hongrun QIAN
%A Jinrong MA
%A Magdalena PAOLINO
%A Zhiying ZHANG
%J Journal of Zhejiang University SCIENCE B
%V 23
%N 2
%P 141-152
%@ 1673-1581
%D 2022
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B2100196

T1 - A high-efficiency and versatile CRISPR/Cas9-mediated HDR-based biallelic editing system
A1 - Xinyi LI
A1 - Bing SUN
A1 - Hongrun QIAN
A1 - Jinrong MA
A1 - Magdalena PAOLINO
A1 - Zhiying ZHANG
J0 - Journal of Zhejiang University Science B
VL - 23
IS - 2
SP - 141
EP - 152
%@ 1673-1581
Y1 - 2022
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B2100196

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 composition. In this technology, the introduced double-strand breaks (DSBs) are mainly repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways. The high-fidelity HDR pathway is used for genome modification, which can introduce artificially controllable insertions, deletions, or substitutions carried by the donor templates. Although high-level knock-out can be easily achieved by NHEJ, accurate HDR-mediated knock-in remains a technical challenge. In most circumstances, although both alleles are broken by endonucleases, only one can be repaired by HDR, and the other one is usually recombined by NHEJ. For gene function studies or disease model establishment, biallelic editing to generate homozygous cell lines and homozygotes is needed to ensure consistent phenotypes. Thus, there is an urgent need for an efficient biallelic editing system. Here, we developed three pairs of integrated selection systems, where each of the two selection cassettes contained one drug-screening gene and one fluorescent marker. Flanked by homologous arms containing the mutated sequences, the selection cassettes were integrated into the target site, mediated by CRISPR/Cas9-induced HDR. Positively targeted cell clones were massively enriched by fluorescent microscopy after screening for drug resistance. We 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 optimization. Our 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.




Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article


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