Full Text:   <3090>

Summary:  <597>

Suppl. Mater.: 

CLC number: 

On-line Access: 2022-02-17

Received: 2021-02-27

Revision Accepted: 2021-06-20

Crosschecked: 0000-00-00

Cited: 0

Clicked: 4774

Citations:  Bibtex RefMan EndNote GB/T7714


Zhiying ZHANG


-   Go to

Article info.
Open peer comments

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.

@article{title="A high-efficiency and versatile CRISPR/Cas9-mediated HDR-based biallelic editing system",
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


[1]AnzaloneAV, RandolphPB, DavisJR, et al., 2019. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature, 576(7785):149-157.

[2]BétermierM, BertrandP, LopezBS, 2014. Is non-homologous end-joining really an inherently error-prone process? PLoS Genet, 10(1):e1004086.

[3]BibikovaM, BeumerK, TrautmanJK, et al., 2003. Enhancing gene targeting with designed zinc finger nucleases. Science, 300(5620):764.

[4]BochJ, ScholzeH, SchornackS, et al., 2009. Breaking the code of DNA binding specificity of TAL-type III effectors. Science, 326(5959):1509-1512.

[5]ChenXY, JanssenJM, LiuJ, et al., 2017. In trans paired nicking triggers seamless genome editing without double-stranded DNA cutting. Nat Commun, 8:657.

[6]ChuVT, WeberT, WefersB, et al., 2015. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol, 33(5):543-548.

[7]CodnerGF, MiannéJ, CaulderA, et al., 2018. Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants. BMC Biol, 16:70.

[8]CongL, RanFA, CoxD, et al., 2013. Multiplex genome engineering using CRISPR/Cas systems. Science, 339(6121):819-823.

[9]EggenschwilerR, MoslemM, FráguasMS, et al., 2016. Improved bi-allelic modification of a transcriptionally silent locus in patient-derived iPSC by Cas9 nickase. Sci Rep, 6:38198.

[10]EvanG, 2012. Taking a back door to target Myc. Science, 335(6066):293-294.

[11]FrangoulH, AltshulerD, CappelliniMD, et al., 2021. CRISPR-Cas9 gene editing for sickle cell disease and β-thalassemia. N Engl J Med, 384(3):252-260.

[12]HsuPD, LanderES, ZhangF, 2014. Development and applications of CRISPR-Cas9 for genome engineering. Cell, 157(6):1262-1278.

[13]JinekM, ChylinskiK, FonfaraI, et al., 2012. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 337(6096):816-821.

[14]KochB, NijmeijerB, KueblbeckM, et al., 2018. Generation and validation of homozygous fluorescent knock-in cells using CRISPR-Cas9 genome editing. Nat Protoc, 13(6):1465-1487.

[15]LiC, BrantE, BudakH, et al., 2021. CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 22(4):253-284.

[16]LiXY, BaiYC, ChengXZ, et al., 2018. Efficient SSA-mediated precise genome editing using CRISPR/Cas9. FEBS J, 285(18):3362-3375.

[17]MaliP, YangLH, EsveltKM, et al., 2013. RNA-guided human genome engineering via Cas9. Science, 339(6121): 823-826.

[18]PaquetD, KwartD, ChenA, et al., 2016. Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature, 533(7601):125-129.

[19]RenCH, XuK, LiuZT, et al., 2015. Dual-reporter surrogate systems for efficient enrichment of genetically modified cells. Cell Mol Life Sci, 72(14):2763-2772.

[20]Saleh-GohariN, HelledayT, 2004. Conservative homologous recombination preferentially repairs DNA double-strand breaks in the S phase of the cell cycle in human cells. Nucleic Acids Res, 32(12):3683-3688.

[21]SavicN, RingnaldaFC, LindsayH, et al., 2018. Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair. eLife, 7:e33761.

[22]ScullyR, PandayA, ElangoR, et al., 2019. DNA double-strand break repair-pathway choice in somatic mammalian cells. Nat Rev Mol Cell Biol, 20(11):698-714.

[23]ShaoSM, RenCH, LiuZT, et al., 2017. Enhancing CRISPR/Cas9-mediated homology-directed repair in mammalian cells by expressing Saccharomyces cerevisiae Rad52. Int J Biochem Cell Biol, 92:43-52.

[24]SurIK, HallikasO, VähärautioA, et al., 2012. Mice lacking a Myc enhancer that includes human SNP rs6983267 are resistant to intestinal tumors. Science, 338(6112):‍1360-1363.

[25]WangG, YangLH, GrishinD, et al., 2017. Efficient, footprint-free human iPSC genome editing by consolidation of Cas9/CRISPR and piggyBac technologies. Nat Protoc, 12(1):88-103.

[26]WuY, XuK, RenCH, et al., 2017. Enhanced CRISPR/Cas9-mediated biallelic genome targeting with dual surrogate reporter-integrated donors. FEBS Lett, 591(6):903-913.

[27]XiLH, SchmidtJC, ZaugAJ, et al., 2015. A novel two-step genome editing strategy with CRISPR-Cas9 provides new insights into telomerase action and TERT gene expression. Genome Biol, 16:231.

[28]YanNN, SunYS, FangYY, et al., 2020. A universal surrogate reporter for efficient enrichment of CRISPR/Cas9-mediated homology-directed repair in mammalian cells. Mol Ther Nucleic Acids, 19:775-789.

[29]YangLH, GuellM, ByrneS, et al., 2013. Optimization of scarless human stem cell genome editing. Nucleic Acids Res, 41(19):9049-9061.

[30]YeL, WangJM, TanYT, et al., 2016. Genome editing using CRISPR-Cas9 to create the HPFH genotype in HSPCs: an approach for treating sickle cell disease and β‍-thalassemia. Proc Natl Acad Sci USA, 113(38):10661-10665.

[31]ZhangBH, 2021. CRISPR/Cas gene therapy. J Cell Physiol, 236(4):2459-2481.

[32]ZhangJP, LiXL, LiGH, et al., 2017. Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage. Genome Biol, 18:35.

[33]ZhaoX, WeiCW, LiJJ, et al., 2017. Cell cycle-dependent control of homologous recombination. Acta Biochim Biophys Sin, 49(8):655-668.

[34]ZuoEW, SunYD, WeiW, et al., 2019. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science, 364(6437):289-292.

Open peer comments: Debate/Discuss/Question/Opinion


Please provide your name, email address and a comment

Journal of Zhejiang University-SCIENCE, 38 Zheda Road, Hangzhou 310027, China
Tel: +86-571-87952783; E-mail: cjzhang@zju.edu.cn
Copyright © 2000 - 2024 Journal of Zhejiang University-SCIENCE