Detection of deleterious on-target effects after HDR-mediated CRISPR editing

SummaryCRISPR genome editing is a promising tool for translational research but can cause undesired editing outcomes, both on-target at the edited locus and off-target at other genomic loci. We investigated the occurrence of deleterious on-target effects in human stem cells after insertion of disease-related mutations by homology-directed repair (HDR). We identified large, mono-allelic genomic deletions and loss-of-heterozygosity that escaped standard quality controls in up to 40% of edited clones. To reliably detect such events, we developed simple, low-cost and universally applicable quantitative genotyping PCR (qgPCR) as well as sequencing-based tools and suggest their usage as additional quality controls after editing. This will help to ensure the integrity of edited loci and increase the reliability of CRISPR editing.

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Use of human stem cells in Huntington disease modeling and translational research

Experimental Neurology ◽

10.1016/j.expneurol.2016.01.021 ◽

2016 ◽

Vol 278 ◽

pp. 76-90 ◽

Cited By ~ 22

Author(s):

Monika M. Golas ◽

Bjoern Sander

Keyword(s):

Stem Cells ◽

Translational Research ◽

Huntington Disease ◽

Disease Modeling ◽

Human Stem Cells

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Controlling homology-directed repair outcomes in human stem cells with dCas9

10.1101/2021.12.16.472942 ◽

2021 ◽

Author(s):

William C Skarnes ◽

Gang Ning ◽

Sofia Giansiracusa ◽

Alexander S Cruz ◽

Cornelis Blauwendraat ◽

...

Keyword(s):

Stem Cells ◽

High Activity ◽

Human Disease ◽

Human Stem Cells ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Homology Directed Repair ◽

Guide Rnas

Modeling human disease in human stem cells requires precise, scarless editing of single nucleotide variants (SNV) on one or both chromosomes. Here we describe improved conditions for Cas9 RNP editing of SNVs that yield high rates of biallelic homology-directed repair. To recover both heterozygous and homozygous SNV clones, catalytically inactive dCas9 was added to moderate high activity Cas9 RNPs. dCas9 can also block re-cutting and damage to SNV alleles engineered with non-overlapping guide RNAs.

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Improving homology-directed repair efficiency in human stem cells

Methods ◽

10.1016/j.ymeth.2019.06.016 ◽

2019 ◽

Vol 164-165 ◽

pp. 18-28 ◽

Cited By ~ 4

Author(s):

William C. Skarnes ◽

Enrica Pellegrino ◽

Justin A. McDonough

Keyword(s):

Stem Cells ◽

Human Stem Cells ◽

Homology Directed Repair ◽

Repair Efficiency

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Hydrogen Sulfide-Releasing Fibrous Membranes: Potential Patches for Stimulating the Human Stem Cells Proliferation and Viability Under Oxidative Stress.

10.20944/preprints201807.0079.v1 ◽

2018 ◽

Author(s):

Ilaria Cacciotti ◽

Matteo Ciocci ◽

Emilia Di Giovanni ◽

Francesca Nanni ◽

Sonia Melino

Keyword(s):

Oxidative Stress ◽

Stem Cells ◽

Hydrogen Sulfide ◽

Tissue Repair ◽

Low Cost ◽

Bioactive Molecules ◽

Poly Lactic Acid ◽

Human Stem Cells ◽

Fibrous Membranes

The design of biomaterial platforms able to release bioactive molecules is mandatory in tissue repair and regenerative medicine. In this context, electrospinning is a user-friendly, versatile and low-cost technique, able to process different kinds of materials in micro- and nano-fibers with a large surface area-to-volume ratio for an optimal release of gaseous signalling molecules. Recently, the antioxidant and anti-inflammatory properties of the endogenous gasotramsmitter hydrogen sulfide (H2S), as well as its ability to stimulate relevant biochemical processes on the growth of mesenchymal stem cells (MSC), have been investigated. Therefore, in this work, new poly(lactic) acid fibrous membranes (PFM), doped and functionalized with H2S slow-releasing donors extracted from garlic, were synthetized. These innovative H2S-releasing mats were characterized for their morphological, thermal, mechanical and biological properties. Their antimicrobial activity and effects on the in vitro human cardiac MSC growth, either in the presence or in the absence of oxidative stress, were here assessed. On the basis of the results here presented, these new H2S-releasing PFM could represent promising and low-cost scaffolds or patches for biomedical applications in tissue repair.

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Hydrogen Sulfide-Releasing Fibrous Membranes: Potential Patches for Stimulating Human Stem Cells Proliferation and Viability under Oxidative Stress

International Journal of Molecular Sciences ◽

10.3390/ijms19082368 ◽

2018 ◽

Vol 19 (8) ◽

pp. 2368 ◽

Cited By ~ 34

Author(s):

Ilaria Cacciotti ◽

Matteo Ciocci ◽

Emilia Di Giovanni ◽

Francesca Nanni ◽

Sonia Melino

Keyword(s):

Oxidative Stress ◽

Stem Cells ◽

Hydrogen Sulfide ◽

Tissue Repair ◽

Low Cost ◽

Bioactive Molecules ◽

Poly Lactic Acid ◽

Human Stem Cells ◽

Fibrous Membranes

The design of biomaterial platforms able to release bioactive molecules is mandatory in tissue repair and regenerative medicine. In this context, electrospinning is a user-friendly, versatile and low-cost technique, able to process different kinds of materials in micro- and nano-fibers with a large surface area-to-volume ratio for an optimal release of gaseous signaling molecules. Recently, the antioxidant and anti-inflammatory properties of the endogenous gasotramsmitter hydrogen sulfide (H2S), as well as its ability to stimulate relevant biochemical processes on the growth of mesenchymal stem cells (MSC), have been investigated. Therefore, in this work, new poly(lactic) acid fibrous membranes (PFM), doped and functionalized with H2S slow-releasing donors extracted from garlic, were synthetized. These innovative H2S-releasing mats were characterized for their morphological, thermal, mechanical, and biological properties. Their antimicrobial activity and effects on the in vitro human cardiac MSC growth, either in the presence or in the absence of oxidative stress, were here assessed. On the basis of the results here presented, these new H2S-releasing PFM could represent promising and low-cost scaffolds or patches for biomedical applications in tissue repair.

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