Improved bi-allelic modification of a transcriptionally silent locus in patient-derived iPSC by Cas9 nickase

Eggenschwiler, Reto; Moslem, Mohsen; Fráguas, Mariane Serra; Galla, Melanie; Papp, Oliver; Naujock, Maximilian; Fonfara, Ines; Gensch, Ingrid; Wähner, Annabell; Beh-Pajooh, Abbas; Mussolino, Claudio; Tauscher, Marcel; Steinemann, Doris; Wegner, Florian; Petri, Susanne; Schambach, Axel; Charpentier, Emmanuelle; Cathomen, Toni; Cantz, Tobias

doi:10.1038/srep38198

Cited by 32 publications

(32 citation statements)

References 34 publications

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“…The use of fluorescent NSM excludes random integration events, enabling clearer sorting gates and isolated biallelic populations. This constitutes an advancement over similar approaches ( Eggenschwiler et al., 2016 ). However, potential limitations are that PSMs could be subjected to position-effect variegation or promotor silencing.…”

Section: Discussionmentioning

confidence: 89%

FACS-Assisted CRISPR-Cas9 Genome Editing Facilitates Parkinson's Disease Modeling

et al. 2017

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SummaryGenome editing and human induced pluripotent stem cells hold great promise for the development of isogenic disease models and the correction of disease-associated mutations for isogenic tissue therapy. CRISPR-Cas9 has emerged as a versatile and simple tool for engineering human cells for such purposes. However, the current protocols to derive genome-edited lines require the screening of a great number of clones to obtain one free of random integration or on-locus non-homologous end joining (NHEJ)-containing alleles. Here, we describe an efficient method to derive biallelic genome-edited populations by the use of fluorescent markers. We call this technique FACS-assisted CRISPR-Cas9 editing (FACE). FACE allows the derivation of correctly edited polyclones carrying a positive selection fluorescent module and the exclusion of non-edited, random integrations and on-target allele NHEJ-containing cells. We derived a set of isogenic lines containing Parkinson's-disease-associated mutations in α-synuclein and present their comparative phenotypes.

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Section: Discussionmentioning

confidence: 89%

FACS-Assisted CRISPR-Cas9 Genome Editing Facilitates Parkinson's Disease Modeling

et al. 2017

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“…These methods necessitate working with a high number of clonal cell lines (typically over a hundred) and can require specialized equipment. Recent PiggyBac transposon methods ( Arias-Fuenzalida et al., 2017 , Eggenschwiler et al., 2016 ) enable complete removal of selectable markers for scarless editing of hPSCs. These methods employ an additional step involving excision of the selectable marker from the edited clones.…”

Section: Introductionmentioning

confidence: 99%

Scarless Genome Editing of Human Pluripotent Stem Cells via Transient Puromycin Selection

Steyer

Cory

et al. 2018

Stem Cell Reports

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SummaryGenome-edited human pluripotent stem cells (hPSCs) have broad applications in disease modeling, drug discovery, and regenerative medicine. We present and characterize a robust method for rapid, scarless introduction or correction of disease-associated variants in hPSCs using CRISPR/Cas9. Utilizing non-integrated plasmid vectors that express a puromycin N-acetyl-transferase (PAC) gene, whose expression and translation is linked to that of Cas9, we transiently select for cells based on their early levels of Cas9 protein. Under optimized conditions, co-delivery with single-stranded donor DNA enabled isolation of clonal cell populations containing both heterozygous and homozygous precise genome edits in as little as 2 weeks without requiring cell sorting or high-throughput sequencing. Edited cells isolated using this method did not contain any detectable off-target mutations and displayed expected functional phenotypes after directed differentiation. We apply the approach to a variety of genomic loci in five hPSC lines cultured using both feeder and feeder-free conditions.

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“…Methods for improving specificity have been developed using either pairs of Cas9 enzymes each of which is unable to generate a DSB alone (Guilinger et al 2014 ; Ran et al 2013 ; Tsai et al 2014 ), truncated guide RNAs (Fu et al 2014 ) or protein engineering of Cas9 to improve specificity (Kleinstiver et al 2016 ; Slaymaker et al 2016 ). Some of these strategies including the double nickase approach (Ran et al 2013 ) have been successfully used in hiPS cells (Eggenschwiler et al 2016 ; Wu et al 2016b ), although these systems will never remove the potential for off-target mutations completely. Therefore at least in the case of disease models, where a small number of lines are produced, it is also possible to sequence the putative off-target sites (or even the whole genome), generate cell lines with independent guides each of which would have a different set of off-targets, or perform another round of genome engineering to repair the introduced genetic change.…”

Section: Genome Editing Methodsmentioning

confidence: 99%

Editing the genome of hiPSC with CRISPR/Cas9: disease models

Bassett

2017

Mamm Genome

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The advent of human-induced pluripotent stem cell (hiPSC) technology has provided a unique opportunity to establish cellular models of disease from individual patients, and to study the effects of the underlying genetic aberrations upon multiple different cell types, many of which would not normally be accessible. Combining this with recent advances in genome editing techniques such as the clustered regularly interspaced short palindromic repeat (CRISPR) system has provided an ability to repair putative causative alleles in patient lines, or introduce disease alleles into a healthy “WT” cell line. This has enabled analysis of isogenic cell pairs that differ in a single genetic change, which allows a thorough assessment of the molecular and cellular phenotypes that result from this abnormality. Importantly, this establishes the true causative lesion, which is often impossible to ascertain from human genetic studies alone. These isogenic cell lines can be used not only to understand the cellular consequences of disease mutations, but also to perform high throughput genetic and pharmacological screens to both understand the underlying pathological mechanisms and to develop novel therapeutic agents to prevent or treat such diseases. In the future, optimising and developing such genetic manipulation technologies may facilitate the provision of cellular or molecular gene therapies, to intervene and ultimately cure many debilitating genetic disorders.

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Improved bi-allelic modification of a transcriptionally silent locus in patient-derived iPSC by Cas9 nickase

Cited by 32 publications

References 34 publications

FACS-Assisted CRISPR-Cas9 Genome Editing Facilitates Parkinson's Disease Modeling

FACS-Assisted CRISPR-Cas9 Genome Editing Facilitates Parkinson's Disease Modeling

Scarless Genome Editing of Human Pluripotent Stem Cells via Transient Puromycin Selection

Editing the genome of hiPSC with CRISPR/Cas9: disease models

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