20 February 2017
New publication in Nature Protocols
This publication, which was co-authored by Yoshiki Narimatsu, Zhang Yang, Henrik Clausen, Hans Wandall and Eric Bennett from CCG, describes how the efficiency of nuclease-mediated genome editing can be evaluated and increased by the sequential use of two techniques: Fluorescence Activated Cell Sorting (FACS) and Indel Detection by Amplicon Analysis (IDAA). FACS enrichment of cells expressing CRISPR/Cas9, TALENs or ZFNs 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, whereas IDAA determines the size and frequency of insertions and deletions elicited by these nucleases in cells, tissues or embryos.
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.
The work was recently described in the UCPH Faculty of Health and Medical Sciences News.
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): Genome editing using FACS enrichment of nuclease-expressing cells and idel detection by amplicon analysis. Nature Protocols 12(3):581-603. doi: 10.1038/nprot.2016.165.