The iCRISPR platform for rapid genome editing in human pluripotent stem cells Journal Article
| Authors: | Zhu, Z.; González, F.; Huangfu, D. |
| Article Title: | The iCRISPR platform for rapid genome editing in human pluripotent stem cells |
| Abstract: | Human pluripotent stem cells (hPSCs) have the potential to generate all adult cell types, including rare or inaccessible human cell populations, thus providing a unique platform for disease studies. To realize this promise, it is essential to develop methods for efficient genetic manipulations in hPSCs. Established using TALEN (transcription activator-like effector nuclease) and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) systems, the iCRISPR platform supports a variety of genome-engineering approaches with high efficiencies. Here, we first describe the establishment of the iCRISPR platform through TALEN-mediated targeting of inducible Cas9 expression cassettes into the AAVS1 locus. Next, we provide a series of technical procedures for using iCRISPR to achieve one-step knockout of one or multiple gene(s), "scarless" introduction of precise nucleotide alterations, as well as inducible knockout during hPSC differentiation. We present an optimized workflow, as well as guidelines for the selection of CRISPR targeting sequences and the design of single-stranded DNA (ssDNA) homology-directed DNA repair templates for the introduction of specific nucleotide alterations. We have successfully used these protocols in four different hPSC lines, including human embryonic stem cells and induced pluripotent stem cells. Once the iCRISPR platform is established, clonal lines with desired genetic modifications can be established in as little as 1 month. The methods described here enable a wide range of genome-engineering applications in hPSCs, thus providing a valuable resource for the creation of diverse hPSC-based disease models with superior speed and ease. © 2014 Elsevier Inc. All rights reserved. |
| Keywords: | controlled study; gene sequence; human cell; nonhuman; mouse; gene targeting; dna repair; gene expression; embryo; embryonic stem cell; gene locus; practice guideline; cell differentiation; genetic manipulation; dna modification; nucleotide sequence; gene inactivation; pluripotent stem cell; single stranded dna; cell clone; nucleotide; gene cassette; dna template; human embryonic stem cells; transcription activator like effector nuclease; human pluripotent stem cells; workflow; crispr/cas; human; priority journal; article; clustered regularly interspaced short palindromic repeat; genome engineering; talens; human pluripotent stem cell |
| Journal Title: | Methods in Enzymology |
| Volume: | 546 |
| ISSN: | 0076-6879 |
| Publisher: | Academic Press |
| Date Published: | 2014-01-01 |
| Start Page: | 215 |
| End Page: | 250 |
| Language: | English |
| DOI: | 10.1016/b978-0-12-801185-0.00011-8 |
| PROVIDER: | scopus |
| PUBMED: | 25398343 |
| PMCID: | PMC4418970 |
| DOI/URL: | |
| Notes: | Chapter 11 in "The Use of CRISPR/Cas9, ZFNs, and TALENs in Generating Site-Specific Genome Alterations" (ISBN: 978-0-12-801185-0) -- Export Date: 4 May 2015 -- Source: Scopus |
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