Genome editing using FACS enrichment of nuclease-expressing cells and indel detection by amplicon analysis

Lonowski LA, Narimatsu Y, Riaz A, Delay CE, Yang Z, Niola F, Duda K, Ober EA, Clausen H, Wandall HH, Hansen SH, Bennett EP, Frödin M (2017)

Publication Type: Journal article

Publication year: 2017

Journal

Nature Protocols Nature Publishing Group

Book Volume: 12

Pages Range: 581-603

Journal Issue: 3

DOI: 10.1038/nprot.2016.165

Abstract

This protocol describes methods for increasing and evaluating the efficiency of genome editing based on the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-Associated 9) system, transcription activator-like effector nucleases (TALENs) or zinc-finger nucleases (ZFNs). First, Indel Detection by Amplicon Analysis (IDAA) determines the size and frequency of insertions and deletions elicited by nucleases in cells, tissues or embryos through analysis of fluorophore-labeled PCR amplicons covering the nuclease target site by capillary electrophoresis in a sequenator. Second, FACS enrichment of cells expressing nucleases linked to fluorescent proteins can be used to maximize knockout or knock-in editing efficiencies or to balance editing efficiency and toxic/off-Target effects. The two methods can be combined to form a pipeline for cell-line editing that facilitates the testing of new nuclease reagents and the generation of edited cell pools or clonal cell lines, reducing the number of clones that need to be generated and increasing the ease with which they are screened. The pipeline shortens the time line, but it most prominently reduces the workload of cell-line editing, which may be completed within 4 weeks.

Involved external institutions

University of Copenhagen DK Denmark (DK) Boston Children's Hospital US United States (USA) (US) Novo Nordisk Foundation / Novo Nordisk Fonden (NNF) DK Denmark (DK)

How to cite

APA:

Lonowski, L.A., Narimatsu, Y., Riaz, A., Delay, C.E., Yang, Z., Niola, F.,... Frödin, M. (2017). Genome editing using FACS enrichment of nuclease-expressing cells and indel detection by amplicon analysis. Nature Protocols, 12(3), 581-603. https://doi.org/10.1038/nprot.2016.165

MLA:

Lonowski, Lindsey A., et al. "Genome editing using FACS enrichment of nuclease-expressing cells and indel detection by amplicon analysis." Nature Protocols 12.3 (2017): 581-603.

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