New Bicistronic TALENs Greatly Improve Genome Editing
José María Martín-Fernández
Karuna Good Cells Technologies SL, Vitoria-Gasteiz, Álava, Spain
Search for more papers by this authorAarne Fleischer
Karuna Good Cells Technologies SL, Vitoria-Gasteiz, Álava, Spain
Consejo Superior de Investigaciones Científicas (CSIC/IMEDEA), Esporles, Spain
Search for more papers by this authorSara Vallejo-Diez
Consejo Superior de Investigaciones Científicas (CSIC/IMEDEA), Esporles, Spain
Search for more papers by this authorEsther Palomino
Consejo Superior de Investigaciones Científicas (CSIC/IMEDEA), Esporles, Spain
Search for more papers by this authorAlmudena Sánchez-Gilabert
Karuna Good Cells Technologies SL, Vitoria-Gasteiz, Álava, Spain
Consejo Superior de Investigaciones Científicas (CSIC/IMEDEA), Esporles, Spain
Search for more papers by this authorRaúl Ruiz
Consejo Superior de Investigaciones Científicas (CSIC/IMEDEA), Esporles, Spain
Search for more papers by this authorYazmine Bejarano
Consejo Superior de Investigaciones Científicas (CSIC/IMEDEA), Esporles, Spain
Current address: Centro de Investigación del Cáncer, Campus Miguel de Unamuno, Salamanca, Spain
Search for more papers by this authorPere Llinàs
Consejo Superior de Investigaciones Científicas (CSIC/IMEDEA), Esporles, Spain
Current address: Josep Carreras Leukaemia Research Institute (IJC), Ctra. de Can Ruti, Camí de les Escoles, Badalona, Spain
Search for more papers by this authorAntoni Gayá
Instituto de Investigación Sanitaria Illes Balears (IDISBA), Fundació Banc de Sang i Teixits de les Illes Balears (FBSTIB), Grupo de Terapia Celular e Ingenieria Tisular, Palma de Mallorca, Spain
Search for more papers by this authorCorresponding Author
Daniel Bachiller
Consejo Superior de Investigaciones Científicas (CSIC/IMEDEA), Esporles, Spain
Corresponding author: [email protected]Search for more papers by this authorJosé María Martín-Fernández
Karuna Good Cells Technologies SL, Vitoria-Gasteiz, Álava, Spain
Search for more papers by this authorAarne Fleischer
Karuna Good Cells Technologies SL, Vitoria-Gasteiz, Álava, Spain
Consejo Superior de Investigaciones Científicas (CSIC/IMEDEA), Esporles, Spain
Search for more papers by this authorSara Vallejo-Diez
Consejo Superior de Investigaciones Científicas (CSIC/IMEDEA), Esporles, Spain
Search for more papers by this authorEsther Palomino
Consejo Superior de Investigaciones Científicas (CSIC/IMEDEA), Esporles, Spain
Search for more papers by this authorAlmudena Sánchez-Gilabert
Karuna Good Cells Technologies SL, Vitoria-Gasteiz, Álava, Spain
Consejo Superior de Investigaciones Científicas (CSIC/IMEDEA), Esporles, Spain
Search for more papers by this authorRaúl Ruiz
Consejo Superior de Investigaciones Científicas (CSIC/IMEDEA), Esporles, Spain
Search for more papers by this authorYazmine Bejarano
Consejo Superior de Investigaciones Científicas (CSIC/IMEDEA), Esporles, Spain
Current address: Centro de Investigación del Cáncer, Campus Miguel de Unamuno, Salamanca, Spain
Search for more papers by this authorPere Llinàs
Consejo Superior de Investigaciones Científicas (CSIC/IMEDEA), Esporles, Spain
Current address: Josep Carreras Leukaemia Research Institute (IJC), Ctra. de Can Ruti, Camí de les Escoles, Badalona, Spain
Search for more papers by this authorAntoni Gayá
Instituto de Investigación Sanitaria Illes Balears (IDISBA), Fundació Banc de Sang i Teixits de les Illes Balears (FBSTIB), Grupo de Terapia Celular e Ingenieria Tisular, Palma de Mallorca, Spain
Search for more papers by this authorCorresponding Author
Daniel Bachiller
Consejo Superior de Investigaciones Científicas (CSIC/IMEDEA), Esporles, Spain
Corresponding author: [email protected]Search for more papers by this authorAbstract
Genome editing has become one of the most powerful tools in present-day stem cell and regenerative medicine research, but despite its rapid acceptance and widespread use, some elements of the technology still need improvement. In this unit, we present data regarding the use of a new, more efficient type of transcription activator-like effector nuclease (TALEN) for gene editing. Our group has generated bicistronic genes in which classical TALEN coding sequences are linked by 2A elements to different reporter molecules, such as fluorochromes (TALEN-F) or membrane receptors (TALEN-M). This structure results in two proteins transcribed from the same transcript, of which the second (the reporter) can be used as the target for selection by fluorescence-assisted cell sorting (FACS) or magnetic-activated cell sorting (MACS). The application of these new TALEN genes allows a rapid enrichment of cells in which both members of the TALEN pair are active, thus eliminating the need for lengthy selection in culture and laborious characterization of a large number of clones. © 2020 by John Wiley & Sons, Inc.
Basic Protocol 1: Generation of new TALENs
Basic Protocol 2: Genome editing using TALEN-F
Alternate Protocol 1: Generation of TALEN-M
Support Protocol 1: mRNA in vitro transcription (IVT) of TALEN-T2A-reporter expression vector
Alternate Protocol 2: Editing of primary T cells using TALEN-M
Basic Protocol 3: Verifying gene editing
Support Protocol 2: Rapid expansion protocol for edited T-cells
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