A pre-screening FISH-based method to detect CRISPR/Cas9 off-targets in mouse embryonic stem cells

Paulis, Marianna; Castelli, Alessandra; Lizier, Michela; Susani, Lucia; Lucchini, Franco; Villa, Anna; Vezzoni, Paolo

doi:10.1038/srep12327

Cited by 22 publications

(15 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Several methods based on deep sequencing, including GUIDE-Seq , digenome-seq , BLESS (Crosetto et al 2013), and HTGTS (Frock et al 2015), have been developed to detect de novo mutations on a genome-wide scale. When CRISPR/Cas9 is used to integrate exogenous DNA constructs, fluorescent in situ hybridization on metaphase chromosomes can also be performed (Paulis et al 2015). They all confirm the existence of off-target mutations, whose locations were not always predicted, but also indicate that the frequency of these events is low in most experimental applications.…”

Section: The Off-target Problemmentioning

confidence: 76%

CRISPR/Cas9: a breakthrough in generating mouse models for endocrinologists

Markossian¹,

Flamant²

2016

Journal of Molecular Endocrinology

View full text Add to dashboard Cite

CRISPR/Cas9 is a recent development in genome editing which is becoming an indispensable element of the genetic toolbox in mice. It provides outstanding possibilities for targeted modification of the genome, and is often extremely efficient. There are currently two main limitations to in ovo genome editing in mice: the first is mosaicism, which is frequent in founder mice. The second is the difficulty to evaluate the advent of off-target mutations, which often imposes to wait for germline transmission to ensure genetic segregation between wanted and unwanted genetic mutations. However rapid progresses are made, suggesting that these difficulties can be overcome in the near future.

show abstract

Section: The Off-target Problemmentioning

confidence: 76%

CRISPR/Cas9: a breakthrough in generating mouse models for endocrinologists

Markossian¹,

Flamant²

2016

Journal of Molecular Endocrinology

View full text Add to dashboard Cite

show abstract

“…In situ hybridization was performed as previously described. 42 In brief, slides were treated with pepsin (0.004%) at 37 C for 30 s and dehydrated through the ethanol series before denaturation in 70% formamide/2Â SSC. The probes were denaturated at 80 C for 10 min and after a pre-annealing of 20 min at 37 C were applied on the slides.…”

Section: Fishmentioning

confidence: 99%

Chromosome Transplantation: A Possible Approach to Treat Human X-linked Disorders

Paulis

Susani

Castelli

et al. 2020

Molecular Therapy - Methods & Clinical Development

Self Cite

View full text Add to dashboard Cite

Many human genetic diseases are associated with gross mutations such as aneuploidies, deletions, duplications, or inversions. For these "structural" disorders, conventional gene therapy, based on viral vectors and/or on programmable nuclease-mediated homologous recombination, is still unsatisfactory. To correct such disorders, chromosome transplantation (CT), defined as the perfect substitution of an endogenous defective chromosome with an exogenous normal one, could be applied. CT re-establishes a normal diploid cell, leaving no marker of the procedure, as we have recently shown in mouse pluripotent stem cells. To prove the feasibility of the CT approach in human cells, we used human induced pluripotent stem cells (hiPSCs) reprogrammed from Lesch-Nyhan (LN) disease patients, taking advantage of their mutation in the X-linked HPRT gene, making the LN cells selectable and distinguishable from the resistant corrected normal cells. In this study, we demonstrate, for the first time, that CT is feasible in hiPSCs: the normal exogenous X chromosome was first transferred using an improved chromosome transfer system, and the extra sex chromosome was spontaneously lost. These CT cells were functionally corrected and maintained their pluripotency and differentiation capability. By inactivation of the autologous HPRT gene, CT paves the way to the correction of hiPSCs from several X-linked disorders.

show abstract

“…Half of the medium was replaced every 2 days. cKit + precursors were immuno-magnetically purified (MS columns with anti-mouse-CD117 MicroBeads, Miltenyi Biotec) at [14][15][16][17][18] ©AlphaMed Press 2019 STEM CELLS 878 CGD Correction by Chromosome Transplantation days and seeded in complete methylcellulose-based medium (MethoCult GF M3534, Stem Cell Technologies, Vancouver, Canada) with the addition of 20 ng/ml of G-CSF for colonyforming unit (CFU) assays. Myeloid colonies were observed after approximately 10-14 days of culture, and at day 28-32, cells were collected and cytospun (800 rpm for 6 minutes; Shandon Cytospin 4, Thermo Fisher Scientific).…”

Section: Phagocyte Differentiation Protocolmentioning

confidence: 99%

Chromosome Transplantation: Correction of the Chronic Granulomatous Disease Defect in Mouse Induced Pluripotent Stem Cells

et al. 2019

Self Cite

View full text Add to dashboard Cite

In spite of the progress in gene editing achieved in recent years, a subset of genetic diseases involving structural chromosome abnormalities, including aneuploidies, large deletions and complex rearrangements, cannot be treated with conventional gene therapy approaches. We have previously devised a strategy, dubbed chromosome transplantation (CT), to replace an endogenous mutated chromosome with an exogenous normal one. To establish a proof of principle for our approach, we chose as disease model the chronic granulomatous disease (CGD), an X‐linked severe immunodeficiency due to abnormalities in CYBB (GP91) gene, including large genomic deletions. We corrected the gene defect by CT in induced pluripotent stem cells (iPSCs) from a CGD male mouse model. The Hprt gene of the endogenous X chromosome was inactivated by CRISPR/Cas9 technology thus allowing the exploitation of the hypoxanthine–aminopterin–thymidine selection system to introduce a normal donor X chromosome by microcell‐mediated chromosome transfer. X‐transplanted clones were obtained, and diploid XY clones which spontaneously lost the endogenous X chromosome were isolated. These cells were differentiated toward the myeloid lineage, and functional granulocytes producing GP91 protein were obtained. We propose the CT approach to correct iPSCs from patients affected by other X‐linked diseases with large deletions, whose treatment is still unsatisfactory. Stem Cells 2019;37:876–887

show abstract

A pre-screening FISH-based method to detect CRISPR/Cas9 off-targets in mouse embryonic stem cells

Cited by 22 publications

References 25 publications

CRISPR/Cas9: a breakthrough in generating mouse models for endocrinologists

CRISPR/Cas9: a breakthrough in generating mouse models for endocrinologists

Chromosome Transplantation: A Possible Approach to Treat Human X-linked Disorders

Chromosome Transplantation: Correction of the Chronic Granulomatous Disease Defect in Mouse Induced Pluripotent Stem Cells

Contact Info

Product

Resources

About