Pipeline for the Generation and Characterization of Transgenic Human Pluripotent Stem Cells Using the CRISPR/Cas9 Technology

Mianné, Joffrey; Bourguignon, Chloé; Van, Chloé Nguyen; Fieldès, Mathieu; Nasri, Amina; Assou, Saïd; Vos, John De

doi:10.3390/cells9051312

Cited by 7 publications

(8 citation statements)

References 57 publications

(101 reference statements)

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“…Interestingly, hiPSCs, which are notoriously hard to modify [ 29 ], did not display this outcome ( Table 1 , bottom row). We screened 80 clones and only one was isolated with a proper monoallelic deletion.…”

Section: Resultsmentioning

confidence: 99%

Unexpectedly High Levels of Inverted Re-Insertions Using Paired sgRNAs for Genomic Deletions

Blayney

Foster

Jagielowicz

et al. 2020

MPs

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Use of dual sgRNAs is a common CRISPR/Cas9-based strategy for the creation of genetic deletions. The ease of screening combined with a rather high rate of success makes this approach a reliable genome engineering procedure. Recently, a number of studies using CRISPR/Cas9 have revealed unwanted large-scale rearrangements, duplications, inversions or larger-than-expected deletions. Strict quality control measures are required to validate the model system, and this crucially depends on knowing which potential experimental outcomes to expect. Using the dual sgRNA deletion approach, our team discovered high levels of excision, inversion and re-insertion at the site of targeting. We detected those at a variety of genomic loci and in several immortalized cell lines, demonstrating that inverted re-insertions are a common by-product with an overall frequency between 3% and 20%. Our findings imply an inherent danger in the misinterpretation of screening data when using only a single PCR screening. While amplification of the region of interest might classify clones as wild type (WT) based on amplicon size, secondary analyses can discover heterozygous (HET) clones among presumptive WTs, and events deemed as HET clones could potentially be full KO. As such, screening for inverted re-insertions helps in decreasing the number of clones required to obtain a full KO. With this technical note, we want to raise awareness of this phenomenon and suggest implementing a standard secondary PCR while screening for deletions.

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Section: Resultsmentioning

confidence: 99%

Unexpectedly High Levels of Inverted Re-Insertions Using Paired sgRNAs for Genomic Deletions

Blayney

Foster

Jagielowicz

et al. 2020

MPs

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“…With the advancement of human knowledge, the use of second-generation vaccines is on the agenda of researchers, and it is hoped that this problem can be solved in the future with the CRISPR method. The use of CRISPR targeting makes it possible to directly select the desired mutation (26). In this study, it was attempted to apply a genome-editing approach using CRISPR-Cas9 in px459 vector and utilize this system to generate a perception about its adaptability to L. major.…”

Section: Discussionmentioning

confidence: 99%

Construction of PX-LmGP63 Using CRISPR-Cas9 as Primary Goal for GP63 gene Knockout in Leishmania major and Leishmanization

Ebrahimi

Alipour

Azizi

et al. 2021

Jundishapur J Microbiol

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Background: Leishmania is an intracellular protozoan parasite that uses complex methods for destroying the innate immune response in mammalian host macrophage cells. Many factors have been identified that play a role in the severity of the parasite’s pathogenicity. One of the factors is the GP63, which is a group of metalloproteinases that disrupts the signaling mechanism of the host cell. Objectives: The aim of this study was to construct PX-LMGP63 vector through CRISPR-Cas9 for GP63 gene knockout in Leishmania major as a potential method for leishmanization. Methods: A pair of gRNAs were designed based on the mRNA sequence of the GP63. Then annealing primers were cloned into the linearized vector PX-459 and transformed into the DH5ɑ competent cells. Then, PCR assay was performed with gene-specific and vector primers to confirm the colonies. In addition, the constructed plasmid was sequenced for final confirmation. Results: The expected size band of 270 was confirmed by PCR. The plasmid sequence showed that the gRNA789 was ligated in the vector. The created structure was named PX-LMGP63 and will be transfected into the promastigote cell in the next step. Conclusions: Owing to the prevalence of cutaneous Leishman as a public health problem in most countries and the lack of an effective vaccine for leishmaniasis, the use of the CRISPR method may make it possible to achieve an effective vaccine in the future.

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“…The combination of these two groundbreaking technologies has tremendous potential, from disease modeling to stem cell-based therapies. Mianne described a pipeline of strategies to efficiently generate, sub-clone, and characterize CRISPR/Cas9-edited iPSC lines in the function of the introduced mutation (indels, point mutations, insertion of large constructs, and deletions) [ 34 ].…”

Section: Stem Cell Biology and Technologymentioning

confidence: 99%

“…This Special Issue includes both research [ 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 ] and reviews articles [ 10 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 ] which cover wide ranges of stem cell research: adult stem cells, cancer stem cells, pluripotent stem cells, and complex 3D organoid/cell aggregate models [ 26 , 27 , 33 ], with the focuses on stem cell biology/technology [ 10 , 23 , 24 , 25 , 26 , 31 , 32 , 34 ], and stem cell-based disease modeling [ 10 , 27 , 29 , 31 , 33 , 38 , 43 ] and cell therapy [ 24 , 28 , 30 , 32 , 35 , 36 , 37 , 39 , 40 , 41 ].…”

Section: Introductionmentioning

confidence: 99%

“…iPSCs can be generated from diverse patient populations, expanded, and differentiated into a disease-related specific cell types (e.g., neurons and cardiomyocytes) that can be either cultured as two-dimensional (2D) monolayers or included in stem cell-derived organoids, which can then be used as a tool to improve the understanding of disease mechanisms and to test therapeutic interventions [9,21,22]. This Special Issue includes both research [23][24][25][26][27][28][29][30][31][32] and reviews articles [10,[33][34][35][36][37][38][39][40][41][42] which cover wide ranges of stem cell research: adult stem cells, cancer stem cells, pluripotent stem cells, and complex 3D organoid/cell aggregate models [26,27,33], with the focuses on stem cell biology/technology [10,[23][24][25][26]31,32,34], and stem cell-based disease modeling [10,27,29,31,33,38,43] and cell therapy [24,28,…”

Section: Introductionmentioning

confidence: 99%

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Stem Cell-Based Disease Modeling and Cell Therapy

Bai

2020

Cells

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Stem cell science is among the fastest moving fields in biology, with many highly promising directions for translatability. To centralize and contextualize some of the latest developments, this Special Issue presents state-of-the-art research of adult stem cells, induced pluripotent stem cells (iPSCs), and embryonic stem cells as well as cancer stem cells. The studies we include describe efficient differentiation protocols of generation of chondrocytes, adipocytes, and neurons, maturation of iPSC-derived cardiomyocytes and neurons, dynamic characterization of iPSC-derived 3D cerebral organoids, CRISPR/Cas9 genome editing, and non-viral minicircle vector-based gene modification of stem cells. Different applications of stem cells in disease modeling are described as well. This volume also highlights the most recent developments and applications of stem cells in basic science research and disease treatments.

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Pipeline for the Generation and Characterization of Transgenic Human Pluripotent Stem Cells Using the CRISPR/Cas9 Technology

Cited by 7 publications

References 57 publications

Unexpectedly High Levels of Inverted Re-Insertions Using Paired sgRNAs for Genomic Deletions

Unexpectedly High Levels of Inverted Re-Insertions Using Paired sgRNAs for Genomic Deletions

Construction of PX-LmGP63 Using CRISPR-Cas9 as Primary Goal for GP63 gene Knockout in Leishmania major and Leishmanization

Stem Cell-Based Disease Modeling and Cell Therapy

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