Please use this identifier to cite or link to this item: https://hdl.handle.net/10356/148681
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dc.contributor.authorLiu, Ivy Kaiwenen_US
dc.contributor.authorNorfala-Aliah Sutrisnohen_US
dc.contributor.authorWang, Yuanmingen_US
dc.contributor.authorTan, Meng Howen_US
dc.date.accessioned2021-05-04T08:25:18Z-
dc.date.available2021-05-04T08:25:18Z-
dc.date.issued2019-
dc.identifier.citationLiu, I. K., Norfala-Aliah Sutrisnoh, Wang, Y. & Tan, M. H. (2019). Genome editing in mammalian cell lines using CRISPR-Cas. Journal of Visualized Experiments, 2019(146), e59086--. https://dx.doi.org/10.3791/59086en_US
dc.identifier.issn1940-087Xen_US
dc.identifier.urihttps://hdl.handle.net/10356/148681-
dc.description.abstractThe clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been successfully repurposed for genome engineering in many different living organisms. Most commonly, the wildtype CRISPR associated 9 (Cas9) or Cas12a endonuclease is used to cleave specific sites in the genome, after which the DNA double-stranded break is repaired via the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway depending on whether a donor template is absent or present respectively. To date, CRISPR systems from different bacterial species have been shown to be capable of performing genome editing in mammalian cells. However, despite the apparent simplicity of the technology, multiple design parameters need to be considered, which often leave users perplexed about how best to carry out their genome editing experiments. Here, we describe a complete workflow from experimental design to identification of cell clones that carry desired DNA modifications, with the goal of facilitating successful execution of genome editing experiments in mammalian cell lines. We highlight key considerations for users to take note of, including the choice of CRISPR system, the spacer length, and the design of a single-stranded oligodeoxynucleotide (ssODN) donor template. We envision that this workflow will be useful for gene knockout studies, disease modeling efforts, or the generation of reporter cell lines.en_US
dc.description.sponsorshipAgency for Science, Technology and Research (A*STAR)en_US
dc.description.sponsorshipMinistry of Education (MOE)en_US
dc.description.sponsorshipNanyang Technological Universityen_US
dc.description.sponsorshipNational Medical Research Council (NMRC)en_US
dc.description.sponsorshipNational Research Foundation (NRF)en_US
dc.language.isoenen_US
dc.relation1431AFG103en_US
dc.relationOFIRG/0017/2016en_US
dc.relationNRF2013-THE001-046en_US
dc.relationNRF2013-THE001-093en_US
dc.relationRG50/17 (S)en_US
dc.relation.ispartofJournal of Visualized Experimentsen_US
dc.rights© 2019 Journal of Visualized Experiments (JoVE). All rights reserved.en_US
dc.subjectEngineering::Chemical engineeringen_US
dc.titleGenome editing in mammalian cell lines using CRISPR-Casen_US
dc.typeJournal Articleen
dc.contributor.schoolSchool of Chemical and Biomedical Engineeringen_US
dc.contributor.organizationAgency for Science, Technology and Research (A∗STAR)en_US
dc.identifier.doi10.3791/59086-
dc.identifier.pmid31033959-
dc.identifier.scopus2-s2.0-85065423754-
dc.identifier.issue146en_US
dc.identifier.volume2019en_US
dc.identifier.spagee59086-en_US
dc.subject.keywordsGeneticsen_US
dc.subject.keywordsClustered Regularly Interspaced Short Palindromic Repeatsen_US
dc.description.acknowledgementM.H.T. is supported by an Agency for Science Technology and Research’s Joint Council Office grant (1431AFG103), a National Medical Research Council grant (OFIRG/0017/2016), National Research Foundation grants (NRF2013-THE001-046 and NRF2013-THE001-093), a Ministry of Education Tier 1 grant (RG50/17 (S)), a startup grant from Nanyang Technological University, and funds for the International Genetically Engineering Machine (iGEM) competition from Nanyang Technological University.en_US
item.grantfulltextnone-
item.fulltextNo Fulltext-
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