Easy quantification of template-directed CRISPR/Cas9 editing

Brinkman, Eva K.; Kousholt, Arne Nedergaard; Harmsen, Tim; Leemans, C. René; Chen, Tao; Jonkers, Jos; Steensel, Bas van

doi:10.1093/nar/gky164

Cited by 150 publications

(109 citation statements)

References 24 publications

(25 reference statements)

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“…Our approach will allow one experimental workflow to be able to analyze HDR and base editing experiments. ICE offers the benefit of not requiring laborious lab-work to construct a synthetic standard and a third Sanger sequencing, unlike TIDER [3].…”

Section: Discussionmentioning

confidence: 99%

Inference of CRISPR Edits from Sanger Trace Data

Hsiau

Conant

Rossi

et al. 2018

Preprint

317

342

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Efficient precision genome editing requires a quick, quantitative, and inexpensive assay of editing outcomes. Here we present ICE (Inference of CRISPR Edits), which enables robust batch analysis of CRISPR edits using Sanger data. ICE proposes potential editing outcomes for single guide, multiplex editing, base editing, and homology-directed repair experiments and then determines which are supported by the data via regression. Additionally, we develop a score called ICE-D (Discordance) that can provide information on large or unexpected edits. We empirically confirm through over 1,800 edits that the ICE algorithm is robust, reproducible, and can analyze CRISPR experiments within days after transfection. We also confirm that ICE strongly correlates with NGS analysis (Amp-Seq). ICE is an improvement over current analysis tools in that it provides batch analysis, is free to use, and can detect a wider variety of edits. It provides investigators with a reliable editing tool that can significantly expedite CRISPR editing workflows. Our ICE tool is available online at ice.synthego.com and the source code is at github.com/synthego-open/ice

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Section: Discussionmentioning

confidence: 99%

Inference of CRISPR Edits from Sanger Trace Data

Hsiau

Conant

Rossi

et al. 2018

Preprint

317

342

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“…After clonal selection, colonies were tested for loss of AP2α through genotyping and Western blotting. Genotyping analysis was assisted with the web tool TIDE: Tracking of Indels by DEcomposition (46). Western blots Cells were lysed in RIPA buffer [150 mM NaCl, 1% IGEPAL® CA-630, 0.5% of Sodium Deoxycholate, 0.1% sodium dodecylsulfate (SDS), and 50mM Tris] supplemented with protease inhibitor cocktail (Biovision, Milpitas, CA).…”

Section: Methodsmentioning

confidence: 99%

β-catenin-Mediated Wnt Signal Transduction Proceeds Through an Endocytosis-Independent Mechanism

Rim

Kinney

Nusse

2020

Preprint

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The Wnt pathway is a key intercellular signaling cascade that regulates development, tissue homeostasis, and regeneration. However, gaps remain in our understanding of the molecular events that take place between ligand-receptor binding and target gene transcription. Here we used a novel tool for quantitative, real-time assessment of endogenous pathway activation, measured in single cells, to answer an unresolved question in the field – whether receptor endocytosis is required for Wnt signal transduction. We combined knockdown or knockout of essential components of Clathrin-mediated endocytosis with quantitative assessment of Wnt signal transduction in mouse embryonic stem cells (mESCs). Disruption of Clathrin-mediated endocytosis did not affect accumulation and nuclear translocation of β-catenin, as measured by single-cell live imaging of endogenous β-catenin, and subsequent target gene transcription. Disruption of another receptor endocytosis pathway, Caveolin-mediated endocytosis, did not affect Wnt pathway activation either. These results, confirmed in multiple cell lines, suggest that endocytosis is not a general requirement for Wnt signal transduction. We show that off-target effects of a drug used to inhibit endocytosis may be one source of the discrepancy among reports on the role of endocytosis in Wnt signaling.

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“…The dual overlapping Gaa c.1826 gRNA strategy achieved 66.7% on-target editing activity (founder mice positive for any Gaa mutation) and 25% HDR efficiency (founder mice positive for Gaa c.1826dupA mutation) ( Table 2). Following founder mice genotyping, we selected a founder with the lowest levels of mosaicism -as determined by TIDER 16 analysisfor mating and segregation of Gaa c.1826dupA mutation. We successfully generated a homozygous Gaa c.1826dupA knock-in mouse after 2 generations of breeding (Figure 2c).…”

Section: Generation and Characterization Of Gaa C1826dupa Transgenic Micementioning

confidence: 99%

CRISPR-Cas9 generated Pompe knock-in murine model exhibits early-onset hypertrophic cardiomyopathy and skeletal muscle weakness

Huang

Kan

Sandfeld

et al. 2019

Preprint

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The goal of this study is to generate and characterize a knock-in model of Pompe disease (PD) a rare, progressive, fatal disorder primarily affecting the cardiac and musculoskeletal systems. While a murine model of PD exists, it bears a Cre/loxP induced exonic insertion of a neomycin cassette and does not completely recapitulate severe human PD -displaying nonfatal hypertrophic cardiomyopathy only late in its natural history. We therefore designed a CRISPR-Cas9 knock-in system targeting the Gaa gene to introduce the known pathogenic CRIM negative Gaa mutation c.1826insA (p.Y609*).Following optimization of our knock-in strategy in cultured murine myoblasts, we successfully generated a Gaa c1826insA mouse model using a dual sgRNA with ssODN donor template approach. Whole genome sequencing and analysis of the Gaa c1826insA murine model establishes that our system is highly specific for the Gaa c1826 target locus and does not induce any off-target mutations or genomic rearrangements. Next, we examined GAA mRNA transcript, protein expression and enzymatic activity levels in our PD knock-in mice. Gaa c1826insA mice display significantly reduced levels of GAA expression and enzymatic activity relative to wild-type mice.We performed echocardiography on Gaa c1826insA mice to assess cardiac structure and function.Gaa c1826insA mice exhibit early-onset, progressive cardiac hypertrophy as measured by significant increases in left ventricular wall thickness and mass index by 3 months of age. We also conducted functional testsgrip strength, inverted screen, gait analysison Gaa c1826insA mice every 3 months to assess overall motor performance. Gaa c1826insA mice display impaired motor strength and coordination relative to wild-type mice. Altogether, our results demonstrate that the Gaa c1826insA murine model recapitulates human infantile-onset Pompe disease and is better suited for evaluation of therapeutic strategies such as genome correction.

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Easy quantification of template-directed CRISPR/Cas9 editing

Cited by 150 publications

References 24 publications

Inference of CRISPR Edits from Sanger Trace Data

Inference of CRISPR Edits from Sanger Trace Data

β-catenin-Mediated Wnt Signal Transduction Proceeds Through an Endocytosis-Independent Mechanism

CRISPR-Cas9 generated Pompe knock-in murine model exhibits early-onset hypertrophic cardiomyopathy and skeletal muscle weakness

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