Isolation of single-base genome-edited human iPS cells without
antibiotic selection

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Abstract

Precise editing of human genomes in pluripotent stem cells by homology-driven repair of targeted nuclease-induced cleavage has been hindered by the difficulty of isolating rare clones. We developed an efficient method to capture rare mutational events, enabling isolation of mutant lines with single-base substitutions without antibiotic selection. This method facilitates efficient induction or reversion of mutations associated with human disease in isogenic human induced pluripotent stem cells.

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Most cited references 21

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Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting

Tomas Cermak, Erin L. Doyle, Michelle Christian … (2011)

TALENs are important new tools for genome engineering. Fusions of transcription activator-like (TAL) effectors of plant pathogenic Xanthomonas spp. to the FokI nuclease, TALENs bind and cleave DNA in pairs. Binding specificity is determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors. We present a method and reagents for efficiently assembling TALEN constructs with custom repeat arrays. We also describe design guidelines based on naturally occurring TAL effectors and their binding sites. Using software that applies these guidelines, in nine genes from plants, animals and protists, we found candidate cleavage sites on average every 35 bp. Each of 15 sites selected from this set was cleaved in a yeast-based assay with TALEN pairs constructed with our reagents. We used two of the TALEN pairs to mutate HPRT1 in human cells and ADH1 in Arabidopsis thaliana protoplasts. Our reagents include a plasmid construct for making custom TAL effectors and one for TAL effector fusions to additional proteins of interest. Using the former, we constructed de novo a functional analog of AvrHah1 of Xanthomonas gardneri. The complete plasmid set is available through the non-profit repository AddGene and a web-based version of our software is freely accessible online.

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EGFR mutations and lung cancer.

Gilda da Cunha Santos, Frances A Shepherd, Ming Sound Tsao (2011)

Epidermal growth factor receptor (EGFR) is a transmembrane protein with cytoplasmic kinase activity that transduces important growth factor signaling from the extracellular milieu to the cell. Given that more than 60% of non-small cell lung carcinomas (NSCLCs) express EGFR, EGFR has become an important therapeutic target for the treatment of these tumors. Inhibitors that target the kinase domain of EGFR have been developed and are clinically active. More importantly, such tyrosine kinase inhibitors (TKIs) are especially effective in patients whose tumors harbor activating mutations in the tyrosine kinase domain of the EGFR gene. More recent trials have suggested that for advanced NSCLC patients with EGFR mutant tumors, initial therapy with a TKI instead of chemotherapy may be the best choice of treatment. Therefore, mutation testing is mandatory to identify these patients, given that selection based only on clinico-pathologic characteristics is inadequate. We review the role of EGFR mutations in the diagnosis and management of NSCLC.

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TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction

Erin L. Doyle, Nicholas J. Booher, Daniel S Standage … (2012)

Transcription activator-like (TAL) effectors are repeat-containing proteins used by plant pathogenic bacteria to manipulate host gene expression. Repeats are polymorphic and individually specify single nucleotides in the DNA target, with some degeneracy. A TAL effector-nucleotide binding code that links repeat type to specified nucleotide enables prediction of genomic binding sites for TAL effectors and customization of TAL effectors for use in DNA targeting, in particular as custom transcription factors for engineered gene regulation and as site-specific nucleases for genome editing. We have developed a suite of web-based tools called TAL Effector-Nucleotide Targeter 2.0 (TALE-NT 2.0; https://boglab.plp.iastate.edu/) that enables design of custom TAL effector repeat arrays for desired targets and prediction of TAL effector binding sites, ranked by likelihood, in a genome, promoterome or other sequence of interest. Search parameters can be set by the user to work with any TAL effector or TAL effector nuclease architecture. Applications range from designing highly specific DNA targeting tools and identifying potential off-target sites to predicting effector targets important in plant disease.

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Author and article information

Journal

Journal ID (nlm-journal-id): 101215604

Journal ID (pubmed-jr-id): 32338

Journal ID (nlm-ta): Nat Methods

Journal ID (iso-abbrev): Nat. Methods

Title: Nature methods

ISSN (Print): 1548-7091

ISSN (Electronic): 1548-7105

Publication date Nihms-submitted: 28 March 2014

Publication date (Electronic): 09 February 2014

Publication date (Print): March 2014

Publication date PMC-release: 01 September 2014

Volume: 11

Issue: 3

Pages: 291-293

Affiliations

[1 ]Gladstone Institute of Cardiovascular Disease, San Francisco, California 94158, USA

[2 ]Department of Pediatrics, University of California San Francisco, San Francisco, California 94143, USA

[3 ]Department of Neurology, University of California San Francisco, San Francisco, California 94158, USA

[4 ]Department of Medicine, University of California San Francisco, San Francisco, California 94143, USA

[5 ]Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, California 94158, USA

Author notes

Correspondence should be addressed to B.R.C. ( bconklin@ 123456gladstone.ucsf.edu )

Article

Manuscript ID: NIHMS556880

DOI: 10.1038/nmeth.2840

PMC ID: 4063274

PubMed ID: 24509632

SO-VID: f1648ad0-0322-4e13-b0d2-4d964e30c55e

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Isolation of single-base genome-edited human iPS cells without antibiotic selection

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Abstract

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Universal stem cells

Most cited references 21

Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting

EGFR mutations and lung cancer.

TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction

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