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

TY - JOUR

T1 - Genome editing using FACS enrichment of nuclease-expressing cells and indel detection by amplicon analysis

AU - Lonowski, Lindsey A

AU - Narimatsu, Yoshiki

AU - Riaz, Anjum

AU - Delay, Catherine Marie Elander

AU - Yang, Zhang

AU - Niola, Francesco

AU - Duda, Katarzyna

AU - Ober, Elke A

AU - Clausen, Henrik

AU - Wandall, Hans H

AU - Hansen, Steen H

AU - Bennett, Eric P

AU - Frödin, Morten

PY - 2017/3/1

Y1 - 2017/3/1

N2 - 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.

AB - 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.

U2 - 10.1038/nprot.2016.165

DO - 10.1038/nprot.2016.165

M3 - Journal article

C2 - 28207001

SN - 1754-2189

VL - 12

SP - 581

EP - 603

JO - Nature Protocols

JF - Nature Protocols

IS - 3

ER -