@article{27e99faaaef9427f8e896820e399f047,
title = "Multiplex Genome Editing of Human Pluripotent Stem Cells Using Cpf1",
abstract = "Targeted genome editing of human pluripotent stem cells (hPSCs) is critical for basic and translational research and can be achieved with site-specific endonucleases. Cpf1 (CRISPR from Prevotella and Francisella) is a programmable DNA endonuclease with AT-rich PAM sequences. In this protocol, we describe procedures for using a single vector system to deliver Cpf1 and CRISPR RNA (crRNA) for genome editing in hPSCs. This protocol enables indel formation and homologous recombination-mediated precise editing at multiple loci. With the delivery of Cpf1 and a single U6 promoter-driven guide RNA array composed of an AAVS1-targeting and a MAFB-targeting crRNA array, efficient multiplex genome editing at the AAVS1 (knockin) and MAFB (knockout) loci in hPSCs could be achieved in a single experiment. The edited hPSCs expressed pluripotency markers and could differentiate into neurons in vitro. This system also generated INS reporter hPSCs with a 6 kb cassette knockin at the INS locus. The INS reporter cells can differentiate into β-cells that express tdTomato and luciferase, permitting fluorescence-activated cell sorting of hPSC-β-cells. By targeted screening of potential off-target sequences that are most homologous to crRNA sequences, no off-target mutations were detected in any of the tested sequences. This work provides an efficient and flexible system for precise genome editing in mammalian cells including hPSCs with the benefits of less off-target effects.",
keywords = "CRISPR, Cpf1, Differentiation of hPSCs, Genome editing, Human pluripotent stem cells (hPSCs), Regenerative medicine",
author = "Haiting Ma",
note = "The authors wish to thank Prof. Rudolf Jaenisch and members of Prof. Jaenisch's laboratory for advice and discussion, Prof. Feng Zhang's laboratory for sharing pX458 and pY010 plasmid through Addgene, Dr. Huan Yang of Prof. Hongkui Deng's laboratory for sharing INS targeting construct, Patrick Autissier, Eleanor Kincaid, and Hanna Aharonov of FACS core laboratory of Whitehead Institute for sorting cells, Gary Rogers of the Whitehead Institute for assistant with medium preparation, the Center for Computational and Integrative Biology (CCIB) at Massachusetts General Hospital for the use of the CCIB DNA Core Facility (Cambridge, MA), which provided NGS and data analysis service, and Arend Vischer of the Ma's laboratory for feedback on this work. This study was supported by a generous gift from Liliana and Hillel Bachrach and in part by the NIH (RO1-CA084198) to R.J., and the University of Illinois start-up funding to H.M. The authors wish to thank Prof. Rudolf Jaenisch and members of Prof. Jaenisch\textbackslash{}u2019s laboratory for advice and discussion, Prof. Feng Zhang\textbackslash{}u2019s laboratory for sharing pX458 and pY010 plasmid through Addgene, Dr. Huan Yang of Prof. Hongkui Deng\textbackslash{}u2019s laboratory for sharing INS targeting construct, Patrick Autissier, Eleanor Kincaid, and Hanna Aharonov of FACS core laboratory of Whitehead Institute for sorting cells, Gary Rogers of the Whitehead Institute for assistant with medium preparation, the Center for Computational and Integrative Biology (CCIB) at Massachusetts General Hospital for the use of the CCIB DNA Core Facility (Cambridge, MA), which provided NGS and data analysis service, and Arend Vischer of the Ma\textbackslash{}u2019s laboratory for feedback on this work. This study was supported by a generous gift from Liliana and Hillel Bachrach and in part by the NIH (RO1-CA084198) to R.J., and the University of Illinois start-up funding to H.M.",
year = "2024",
month = nov,
day = "20",
doi = "10.21769/BioProtoc.5108",
language = "English (US)",
volume = "14",
journal = "Bio-protocol",
issn = "2331-8325",
publisher = "Bio-protocol LLC",
number = "22",
}