Rapid screening of single guide RNA targeting pig genome and the harvesting of monoclonal cells by microarray seal_Journal of Biomedical Engineering

Authors：

GAO Mengyu ^1,2 , ZHU Xinglong ^2,3 , WANG Shisheng ⁴ , ZHANG Bingqi ^2,3 , ZHANG Yunlin ^2,3 , HE Yuting ^2,3 , ZHOU Yanyan ² , LI Shun ⁵ , YANG guang ⁶ , LIAO Guangneng ⁶ ,  BAO Ji ^2,3 ,  BU Hong ^1,2

1. Department of Pathology, West China Hospital, Sichuan University, Chengdu 610041, P.R.China;
2. Institute of Clinical Pathology, West China Hospital, Sichuan University, Chengdu 610041, P.R.China;
3. Laboratory of Pathology, Key Laboratory of Transplant Engineering and Immunology, NHFPC; West China Hospital, Sichuan University, Chengdu 610041, P.R.China;
4. West China - Washington Mitochondria and Metabolism Research Center, West China Hospital, Sichuan University, Chengdu 610041, P.R.China;
5. Department of Biophysics, School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 611731, P.R.China;
6. Experimental Animal Center, West China Hospital, Sichuan University, Chengdu 610041, P.R.China;

Corresponding author：

BAO Ji, Email: baoji@scu.edu.cn; BU Hong, Email: hongbu@scu.edu.cn

Keywords：

regular short repetitive Palindromic sequence clusters and CRISPR- associated proteins 9; gene editing for large animals; fumarylacetoacetate hydrolase gene knockout; rapid screening of sgRNA; patterned microarray

DOI：

10.7507/1001-5515.202006032

Video：

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Abstract Full text Figures/Tables Video References Cited by

The emergence of regular short repetitive palindromic sequence clusters (CRISPR) and CRISPR- associated proteins 9 (Cas9) gene editing technology has greatly promoted the wide application of genetically modified pigs. Efficient single guide RNA (sgRNA) is the key to the success of gene editing using CRISPR/Cas9 technology. For large animals with a long reproductive cycle, such as pigs, it is necessary to screen out efficient sgRNA in vitro to avoid wasting time and resource costs before animal experiments. In addition, how to efficiently obtain positive gene editing monoclonal cells is a difficult problem to be solved. In this study, a rapid sgRNA screening method targeting the pig genome was established and we rapidly obtained Fah gene edited cells, laying a foundation for the subsequent production of Fah knockout pigs as human hepatocyte bioreactor. At the same time, the method of obtaining monoclonal cells using pattern microarray culture technology was explored.

Citation： GAO Mengyu, ZHU Xinglong, WANG Shisheng, ZHANG Bingqi, ZHANG Yunlin, HE Yuting, ZHOU Yanyan, LI Shun, YANG guang, LIAO Guangneng, BAO Ji, BU Hong. Rapid screening of single guide RNA targeting pig genome and the harvesting of monoclonal cells by microarray seal. Journal of Biomedical Engineering, 2021, 38(1): 111-121. doi: 10.7507/1001-5515.202006032 Copy

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2.	Yu W, Mookherjee S, Chaitankar V, et al. Nrl knockdown by AAV-delivered CRISPR/Cas9 prevents retinal degeneration in mice. Nat Commun, 2017, 8: 14716.
3.	Song Jun, Yang Dongshan, Xu Jie, et al. RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency. Nat Commun, 2016, 7: 10548.
4.	Yan S, Tu Z, Liu Z, et al. A huntingtin knockin pig model recapitulates features of selective neurodegeneration in huntington's disease. Cell, 2018, 173(4): 989-1002, e13.
5.	Yao X, Liu Z, Wang X, et al. Generation of knock-in cynomolgus monkey via CRISPR/Cas9 editing. Cell Res, 2018, 28(3): 379-382.
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13.	Zhou Xiaoqing, Xin Jige, Fan Nana, et al. Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer. Cellular and Molecular Life Sciences, 2015, 72(6): 1175-1184.
14.	Yang R, Lemaître V, Huang C, et al. Monoclonal cell line generation and Crispr/Cas9 manipulation via single-cell electroporation. Small, 2018, 14(12): e1702495.
15.	Zhan T, Rindtorff N, Betge J, et al. CRISPR/Cas9 for cancer research and therapy. Semin Cancer Biol, 2019, 55: 106-119.
16.	Shalem O, Sanjana N E, Hartenian E, et al. Genome-Scale CRISPR-Cas9 knockout screening in human cells. Science, 2014, 343(6166): 84-87.
17.	Sanjana N E, Shalem O, Zhang Feng. Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods, 2014, 11(8): 783-784.
18.	Kato-Inui T, Takahashi G, Hsu S, et al. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 with improved proof-reading enhances homology-directed repair. Nucleic Acids Res, 2018, 46(9): 4677-4688.
19.	Zheng C X, Wang S M, Bai Y H, et al. Lentiviral vectors and Adeno-associated virus vectors: useful tools for gene transfer in pain research. Anat Rec (Hoboken), 2018, 301(5): 825-836.
20.	Lino C A, Harper J C, Carney J P, et al. Delivering CRISPR: a review of the challenges and approaches. Drug Deliv, 2018, 25(1): 1234-1257.
21.	Jiang D J, Xu C L, Tsang S H. Revolution in gene medicine therapy and genome surgery. Genes (Basel), 2018, 9(12): 575.
22.	Liang Xiquan, Potter J, Kumar S, et al. Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease, and donor DNA. J Biotechnol, 2017, 241: 136-146.
23.	Zhang J H, Adikaram P, Pandey M, et al. Optimization of genome editing through CRISPR-Cas9 engineering. Bioengineered, 2016, 7(3): 166-174.
24.	Liu C, Zhang L, Liu H, et al. Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications. J Control Release, 2017, 266: 17-26.
25.	Kim H, Um E, Cho S R, et al. Surrogate reporters for enrichment of cells with nuclease-induced mutations. Nat Methods, 2011, 8(11): 941-943.
26.	Ramakrishna S, Cho S W, Kim S, et al. Surrogate reporter-based enrichment of cells containing RNA-guided Cas9 nuclease-induced mutations. Nat Commun, 2014, 5: 3378.
27.	Ren C, Xu K, Liu Z, et al. Dual-reporter surrogate systems for efficient enrichment of genetically modified cells. Cell Mol Life Sci, 2015, 72(14): 2763-2772.
28.	Li Y, Park A I, Mou H, et al. A versatile reporter system for CRISPR-mediated chromosomal rearrangements. Genome Biol, 2015, 16(1): 111.
29.	Grompe M. Fah knockout animals as models for therapeutic liver repopulation. Adv Exp Med Biol, 2017, 959: 215-230.
30.	Nicolas C T, Hickey R D, Allen K L, et al. Hepatocyte spheroids as an alternative to single cells for transplantation after ex vivo gene therapy in mice and pig models. Surgery, 2018, 164(3): 473-481.

1. Liang P, Sun H, Zhang X, et al. Effective and precise adenine base editing in mouse zygotes. Protein Cell, 2018, 9(9): 808-813.
2. Yu W, Mookherjee S, Chaitankar V, et al. Nrl knockdown by AAV-delivered CRISPR/Cas9 prevents retinal degeneration in mice. Nat Commun, 2017, 8: 14716.
3. Song Jun, Yang Dongshan, Xu Jie, et al. RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency. Nat Commun, 2016, 7: 10548.
4. Yan S, Tu Z, Liu Z, et al. A huntingtin knockin pig model recapitulates features of selective neurodegeneration in huntington's disease. Cell, 2018, 173(4): 989-1002, e13.
5. Yao X, Liu Z, Wang X, et al. Generation of knock-in cynomolgus monkey via CRISPR/Cas9 editing. Cell Res, 2018, 28(3): 379-382.
6. Cui Y, Niu Y, Zhou J, et al. Generation of a precise Oct4-hrGFP knockin cynomolgus monkey model via CRISPR/Cas9-assisted homologous recombination. Cell Res, 2018, 28(3): 383-386.
7. Kang J T, Cho B, Ryu J, et al. Biallelic modification of IL2RG leads to severe combined immunodeficiency in pigs. Reprod Biol Endocrinol, 2016, 14(1): 74.
8. Kang J T, Ryu J, Cho B, et al. Generation of RUNX3 knockout pigs using CRISPR/Cas9-mediated gene targeting. Reproduction in Domestic Animals, 2016, 51(6): 970-978.
9. Holm I E, Alstrup A K, Luo Y. Genetically modified pig models for neurodegenerative disorders. J Pathol, 2016, 238(2): 267-287.
10. Song Chanwoo, Lee J, Lee S Y. Genome engineering and gene expression control for bacterial strain development. Biotechnol J, 2015, 10(1): 56-68.
11. Amitai G, Sorek R. CRISPR-Cas adaptation: insights into the mechanism of action. Nat Rev Microbiol, 2016, 14(2): 67-76.
12. Yu H, Long W, Zhang X, et al. Generation of GHR-modified pigs as Laron syndrome models via a dual-sgRNAs/Cas9 system and somatic cell nuclear transfer. J Transl Med, 2018, 16(1): 41.
13. Zhou Xiaoqing, Xin Jige, Fan Nana, et al. Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer. Cellular and Molecular Life Sciences, 2015, 72(6): 1175-1184.
14. Yang R, Lemaître V, Huang C, et al. Monoclonal cell line generation and Crispr/Cas9 manipulation via single-cell electroporation. Small, 2018, 14(12): e1702495.
15. Zhan T, Rindtorff N, Betge J, et al. CRISPR/Cas9 for cancer research and therapy. Semin Cancer Biol, 2019, 55: 106-119.
16. Shalem O, Sanjana N E, Hartenian E, et al. Genome-Scale CRISPR-Cas9 knockout screening in human cells. Science, 2014, 343(6166): 84-87.
17. Sanjana N E, Shalem O, Zhang Feng. Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods, 2014, 11(8): 783-784.
18. Kato-Inui T, Takahashi G, Hsu S, et al. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 with improved proof-reading enhances homology-directed repair. Nucleic Acids Res, 2018, 46(9): 4677-4688.
19. Zheng C X, Wang S M, Bai Y H, et al. Lentiviral vectors and Adeno-associated virus vectors: useful tools for gene transfer in pain research. Anat Rec (Hoboken), 2018, 301(5): 825-836.
20. Lino C A, Harper J C, Carney J P, et al. Delivering CRISPR: a review of the challenges and approaches. Drug Deliv, 2018, 25(1): 1234-1257.
21. Jiang D J, Xu C L, Tsang S H. Revolution in gene medicine therapy and genome surgery. Genes (Basel), 2018, 9(12): 575.
22. Liang Xiquan, Potter J, Kumar S, et al. Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease, and donor DNA. J Biotechnol, 2017, 241: 136-146.
23. Zhang J H, Adikaram P, Pandey M, et al. Optimization of genome editing through CRISPR-Cas9 engineering. Bioengineered, 2016, 7(3): 166-174.
24. Liu C, Zhang L, Liu H, et al. Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications. J Control Release, 2017, 266: 17-26.
25. Kim H, Um E, Cho S R, et al. Surrogate reporters for enrichment of cells with nuclease-induced mutations. Nat Methods, 2011, 8(11): 941-943.
26. Ramakrishna S, Cho S W, Kim S, et al. Surrogate reporter-based enrichment of cells containing RNA-guided Cas9 nuclease-induced mutations. Nat Commun, 2014, 5: 3378.
27. Ren C, Xu K, Liu Z, et al. Dual-reporter surrogate systems for efficient enrichment of genetically modified cells. Cell Mol Life Sci, 2015, 72(14): 2763-2772.
28. Li Y, Park A I, Mou H, et al. A versatile reporter system for CRISPR-mediated chromosomal rearrangements. Genome Biol, 2015, 16(1): 111.
29. Grompe M. Fah knockout animals as models for therapeutic liver repopulation. Adv Exp Med Biol, 2017, 959: 215-230.
30. Nicolas C T, Hickey R D, Allen K L, et al. Hepatocyte spheroids as an alternative to single cells for transplantation after ex vivo gene therapy in mice and pig models. Surgery, 2018, 164(3): 473-481.

Journal of Biomedical Engineering

Rapid screening of single guide RNA targeting pig genome and the harvesting of monoclonal cells by microarray seal

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