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Title: Genome editing in mammalian cell lines using CRISPR-Cas
Authors: Liu, Ivy Kaiwen
Norfala-Aliah Sutrisnoh
Wang, Yuanming
Tan, Meng How
Keywords: Engineering::Chemical engineering
Issue Date: 2019
Source: Liu, 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--.
Project: 1431AFG103
RG50/17 (S)
Journal: Journal of Visualized Experiments
Abstract: The 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.
ISSN: 1940-087X
DOI: 10.3791/59086
Rights: © 2019 Journal of Visualized Experiments (JoVE). All rights reserved.
Fulltext Permission: none
Fulltext Availability: No Fulltext
Appears in Collections:SCBE Journal Articles

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