Generating CRISPR‐Cas9‐Mediated Null Mutations and Screening Targeting Efficiency in Human Pluripotent Stem Cells

Bower, Oliver J.; McCarthy, Afshan; Lea, Rebecca A.; Alanis-Lobato, Gregorio; Zohren, Jasmin; Gerri, Claudia; Turner, James M. A.; Niakan, Kathy K.

doi:10.1002/cpz1.232

Cited by 2 publications

(1 citation statement)

References 97 publications

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“…In vitro modeling enables us to reveal the role of specific genes and chromosomal aberrations in both the self-renewal and differentiation of TE/TSCs and investigate the molecular basis of pregnancy complications, for which failures of trophoblast growth and differentiation could be underlying causes. This technology could be greatly advanced by integrating it with the possibility of the genetic manipulation of human stem cells [ 233 ] as well as human embryos [ 234 , 235 , 236 ]. In addition, methods are being developed for the correction of large-scale chromosomal abnormalities, including CNVs and numerical aberrations, in PSCs [ 237 , 238 ].…”

Section: Future Perspectivesmentioning

confidence: 99%

Stem Cell-Based Trophoblast Models to Unravel the Genetic Causes of Human Miscarriages

Nikitina

Lebedev

2022

Cells

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Miscarriage affects approximately 15% of clinically recognized pregnancies, and 1–3% of couples experience pregnancy loss recurrently. Approximately 50–60% of miscarriages result from chromosomal abnormalities, whereas up to 60% of euploid recurrent abortions harbor variants in candidate genes. The growing number of detected genetic variants requires an investigation into their role in adverse pregnancy outcomes. Since placental defects are the main cause of first-trimester miscarriages, the purpose of this review is to provide a survey of state-of-the-art human in vitro trophoblast models that can be used for the functional assessment of specific abnormalities/variants implicated in pregnancy loss. Since 2018, when primary human trophoblast stem cells were first derived, there has been rapid growth in models of trophoblast lineage. It has been found that a proper balance between self-renewal and differentiation in trophoblast progenitors is crucial for the maintenance of pregnancy. Different responses to aneuploidy have been shown in human embryonic and extra-embryonic lineages. Stem cell-based models provide a powerful tool to explore the effect of a specific aneuploidy/variant on the fetus through placental development, which is important, from a clinical point of view, for deciding on the suitability of embryos for transfer after preimplantation genetic testing for aneuploidy.

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Section: Future Perspectivesmentioning

confidence: 99%

Stem Cell-Based Trophoblast Models to Unravel the Genetic Causes of Human Miscarriages

Nikitina

Lebedev

2022

Cells

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Semi-automated optimized method to isolate CRISPR/Cas9 edited human pluripotent stem cell clones

Frank

Cailleret

Nelep³

et al. 2023

Stem Cell Res Ther

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Background CRISPR/Cas9 editing systems are currently used to generate mutations in a particular gene to mimic a genetic disorder in vitro. Such “disease in a dish” models based on human pluripotent stem cells (hPSCs) offer the opportunity to have access to virtually all cell types of the human body. However, the generation of mutated hPSCs remains fastidious. Current CRISPR/Cas9 editing approaches lead to a mixed cell population containing simultaneously non-edited and a variety of edited cells. These edited hPSCs need therefore to be isolated through manual dilution cloning, which is time-consuming, labor intensive and tedious. Methods Following CRISPR/Cas9 edition, we obtained a mixed cell population with various edited cells. We then used a semi-automated robotic platform to isolate single cell-derived clones. Results We optimized CRISPR/Cas9 editing to knock out a representative gene and developed a semi-automated method for the clonal isolation of edited hPSCs. This method is faster and more reliable than current manual approaches. Conclusions This novel method of hPSC clonal isolation will greatly improve and upscale the generation of edited hPSCs required for downstream applications including disease modeling and drug screening. Graphical Abstract

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Generating CRISPR‐Cas9‐Mediated Null Mutations and Screening Targeting Efficiency in Human Pluripotent Stem Cells

Cited by 2 publications

References 97 publications

Stem Cell-Based Trophoblast Models to Unravel the Genetic Causes of Human Miscarriages

Stem Cell-Based Trophoblast Models to Unravel the Genetic Causes of Human Miscarriages

Semi-automated optimized method to isolate CRISPR/Cas9 edited human pluripotent stem cell clones

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