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

Miyaoka, Yuichiro; Chan, Amanda H.; Judge, Luke M.; Yoo, Jennie; Huang, Miller; Nguyen, Trieu; Lizarraga, Paweena; So, Po-Lin; Conklin, Bruce R.

doi:10.1038/nmeth.2840

Cited by 280 publications

(299 citation statements)

References 29 publications

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“…These populations were mixed clones, derived from plating 2-10 transfected cells 21 . PCR analysis of the EWS/FLI1 DNA fusion region ( Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

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Engineering human tumour-associated chromosomal translocations with the RNA-guided CRISPR–Cas9 system

et al. 2014

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Cancer-related human chromosomal translocations are generated through the illegitimate joining of two non-homologous chromosomes affected by double-strand breaks (DSB). Effective methodologies to reproduce precise reciprocal tumour-associated chromosomal translocations are required to gain insight into the initiation of leukaemia and sarcomas. Here we present a strategy for generating cancer-related human chromosomal translocations in vitro based on the ability of the RNA-guided CRISPR-Cas9 system to induce DSBs at defined positions. Using this approach we generate human cell lines and primary cells bearing chromosomal translocations resembling those described in acute myeloid leukaemia and Ewing's sarcoma at high frequencies. FISH and molecular analysis at the mRNA and protein levels of the fusion genes involved in these engineered cells reveal the reliability and accuracy of the CRISPR-Cas9 approach, providing a powerful tool for cancer studies.

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“…These populations were mixed clones, derived from plating 2-10 transfected cells 21 . PCR analysis of the EWS/FLI1 DNA fusion region ( Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

“…3). Scoring of the 10 best-fitting potential sequences 21 revealed no off-target mutations (Fig. 2).…”

Section: Resultsmentioning

confidence: 99%

Engineering human tumour-associated chromosomal translocations with the RNA-guided CRISPR–Cas9 system

et al. 2014

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“…This posttranscriptional gene-replacement strategy is simple, rapid to use, and genetically encoded. Recent advances in genome engineering using transcription activator-like endonucleases and CRISPR show great promise for gene ablation studies; however, the ability to use these techniques for site-specific biallelic replacement is not guaranteed and requires a large investment in reagents and screening time (36). We believe the PTGR strategy may be more broadly appropriate to many problems where one wishes to simultaneously replace endogenous biallelic expression with an engineered allele variant and screen for emergent functions.…”

Section: Discussionmentioning

confidence: 99%

Engineered cellular gene-replacement platform for selective and inducible proteolytic profiling

Morgan

Diaz

Zeitlin

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Cellular demolition during apoptosis is completed by executioner caspases, that selectively cleave more than 1,500 proteins but whose individual roles are challenging to assess. Here, we used an optimized site-specific and inducible protease to examine the role of a classic apoptotic node, the caspase-activated DNase (CAD). CAD is activated when caspases cleave its endogenous inhibitor ICAD, resulting in the characteristic DNA laddering of apoptosis. We describe a posttranscriptional gene replacement (PTGR) approach where endogenous biallelic ICAD is knocked down and simultaneously replaced with an engineered allele that is susceptible to inducible cleavage by tobacco etch virus protease. Remarkably, selective activation of CAD alone does not induce cell death, although hallmarks of DNA damage are detected in human cancer cell lines. Our data strongly support that the highly cooperative action of CAD and inhibition of DNA repair systems are critical for the DNA laddering phenotype in apoptosis. Furthermore, the PTGR approach provides a general means for replacing wild-type protein function with a precisely engineered mutant at the transcriptional level that should be useful for cell engineering studies.DNA damage | apoptosis | ICAD | site-specific proteolysis |

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“…Therefore, it is necessary to design experimental procedures for the concentration of ssODN-knock-in cells. To date, sibselection and transient drug selection methods to achieve this concentration have been reported [52,79], but further improvements are needed for practical use.…”

Section: Ssodn-mediated Single Nucleotide Variation (Snv) Knock-inmentioning

confidence: 99%

Updated summary of genome editing technology in human cultured cells linked to human genetics studies

2017

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Current deep-sequencing technology provides a mass of nucleotide variations associated with human genetic disorders to accelerate the identification of causative mutations. To understand the etiology of genetic disorders, reverse genetics in human cultured cells is a useful approach for modeling a disease in vitro. However, gene targeting in human cultured cells is difficult because of their low activity of homologous recombination. Engineered endonucleases enable enhancement of the local activation of DNA repair pathways at the human genome target site to rewrite the desired sequence, thereby efficiently generating disease-modeling cultured cell clones. These edited cells can be used to explore the molecular functions of a causative gene product to uncover the etiological mechanisms. The correction of mutations in patient cells using genome editing technology could contribute to the development of unique gene therapies. This technology can also be applied to screening causative mutations. Rare genetic disorders and non-exonic mutation-caused diseases remain frontier in the field of human genetics as it is difficult to validate whether the extracted nucleotide variants are mutation or polymorphism. When isogenic human cultured cells with a candidate variant reproduce the pathogenic phenotypes, it is confirmed that the variant is a causative mutation.

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

Cited by 280 publications

References 29 publications

Engineering human tumour-associated chromosomal translocations with the RNA-guided CRISPR–Cas9 system

Engineering human tumour-associated chromosomal translocations with the RNA-guided CRISPR–Cas9 system

Engineered cellular gene-replacement platform for selective and inducible proteolytic profiling

Updated summary of genome editing technology in human cultured cells linked to human genetics studies

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