Generation of a dual edited human induced pluripotent stem cell Myl7-GFP reporter line with inducible CRISPRi/dCas9

Temporal regulation of CRISPRi activity is critical for genetic screens. Here, we present an inducible CRISPRi platform enabling selection of iPSC-derived cardiomyocytes and reversible gene knockdown. We targeted a doxycycline-inducible dCas9-KRAB-mCherry cassette into the AAVS1 locus in an MYL7-mGF...

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Detalles Bibliográficos
Autores principales: Metzl-Raz, Eyal, Bharucha, Nike, Ataam, Jennifer Arthur, Gavidia, Alexandra A., Greenleaf, William J., Karakikes, Ioannis
Formato: Online Artículo Texto
Lenguaje:English
Publicado: 2022
Materias:
Article
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9169598/
https://www.ncbi.nlm.nih.gov/pubmed/35325819
http://dx.doi.org/10.1016/j.scr.2022.102754
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author Metzl-Raz, Eyal
Bharucha, Nike
Ataam, Jennifer Arthur
Gavidia, Alexandra A.
Greenleaf, William J.
Karakikes, Ioannis
author_facet Metzl-Raz, Eyal
Bharucha, Nike
Ataam, Jennifer Arthur
Gavidia, Alexandra A.
Greenleaf, William J.
Karakikes, Ioannis
author_sort Metzl-Raz, Eyal
collection PubMed
description Temporal regulation of CRISPRi activity is critical for genetic screens. Here, we present an inducible CRISPRi platform enabling selection of iPSC-derived cardiomyocytes and reversible gene knockdown. We targeted a doxycycline-inducible dCas9-KRAB-mCherry cassette into the AAVS1 locus in an MYL7-mGFP reporter iPSC line. A clone with bi-allelic integration displayed minimally leaky CRISPRi activity and strong expression upon addition of doxycycline in iPSCs, iPSC-derived cardiomyocytes, and multilineage differentiated cells. The CRISPRi activity was validated by targeting the MYOCD gene in iPSC cardiomyocytes. In summary, we developed a robust inducible CRISPRi platform to interrogate gene function in human iPSC-derived cardiomyocytes and other cells.
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spelling pubmed-91695982022-06-06 Generation of a dual edited human induced pluripotent stem cell Myl7-GFP reporter line with inducible CRISPRi/dCas9 Metzl-Raz, Eyal Bharucha, Nike Ataam, Jennifer Arthur Gavidia, Alexandra A. Greenleaf, William J. Karakikes, Ioannis Stem Cell Res Article Temporal regulation of CRISPRi activity is critical for genetic screens. Here, we present an inducible CRISPRi platform enabling selection of iPSC-derived cardiomyocytes and reversible gene knockdown. We targeted a doxycycline-inducible dCas9-KRAB-mCherry cassette into the AAVS1 locus in an MYL7-mGFP reporter iPSC line. A clone with bi-allelic integration displayed minimally leaky CRISPRi activity and strong expression upon addition of doxycycline in iPSCs, iPSC-derived cardiomyocytes, and multilineage differentiated cells. The CRISPRi activity was validated by targeting the MYOCD gene in iPSC cardiomyocytes. In summary, we developed a robust inducible CRISPRi platform to interrogate gene function in human iPSC-derived cardiomyocytes and other cells. 2022-05 2022-03-18 /pmc/articles/PMC9169598/ /pubmed/35325819 http://dx.doi.org/10.1016/j.scr.2022.102754 Text en https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/ (https://creativecommons.org/licenses/by-nc-nd/4.0/) ).
spellingShingle Article
Metzl-Raz, Eyal
Bharucha, Nike
Ataam, Jennifer Arthur
Gavidia, Alexandra A.
Greenleaf, William J.
Karakikes, Ioannis
Generation of a dual edited human induced pluripotent stem cell Myl7-GFP reporter line with inducible CRISPRi/dCas9
title Generation of a dual edited human induced pluripotent stem cell Myl7-GFP reporter line with inducible CRISPRi/dCas9
title_full Generation of a dual edited human induced pluripotent stem cell Myl7-GFP reporter line with inducible CRISPRi/dCas9
title_fullStr Generation of a dual edited human induced pluripotent stem cell Myl7-GFP reporter line with inducible CRISPRi/dCas9
title_full_unstemmed Generation of a dual edited human induced pluripotent stem cell Myl7-GFP reporter line with inducible CRISPRi/dCas9
title_short Generation of a dual edited human induced pluripotent stem cell Myl7-GFP reporter line with inducible CRISPRi/dCas9
title_sort generation of a dual edited human induced pluripotent stem cell myl7-gfp reporter line with inducible crispri/dcas9
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9169598/
https://www.ncbi.nlm.nih.gov/pubmed/35325819
http://dx.doi.org/10.1016/j.scr.2022.102754
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