Aug 2026· Stem Cell Research· Vol 95, pp.
104070
· 0 citations· 4 references
Medicine
TL;DR
The CRISPR/Cas9 system is utilized to generate a homozygous Mt4 knockout (Mt4-/-) mouse embryonic stem cell (mESC) line that maintains normal morphology, pluripotency, and the ability to differentiate into all three germ layers.
Abstract
Metallothionein 4 (MT4) is a low-molecular-weight, cysteine-rich metal-binding protein belonging to the metallothionein family. It exhibits unique skin developmental and differentiation inhibitory activity when functionally impaired and regulates skin cell growth and disease through multiple mechanisms. However, its exact role in cell fate determination remains unknown. Here, we utilized the CRISPR/Cas9 system to generate a homozygous Mt4 knockout (Mt4-/-) mouse embryonic stem cell (mESC) line. This cell line maintains normal morphology, pluripotency, and the ability to differentiate into all three germ layers. It provides a valuable resource for investigating the mechanisms underlying skin diseases caused by MT4 gene mutations.
Step-by-step methods for deploying CRISPR/Cas9 to engineer a TERT-immortalized human epidermal keratinocyte line (N/TERT-2G) to generate monoclonal heterozygous and homozygous knockout cell lines are described.
Anthony Coon, Jessica L. Ayers, Christopher Cole et al.· Methods in molecular biology· 0 citations
A human induced Pluripotent Stem Cell line harboring a doxycycline-inducible Cas9 and an NKX2-1-EGFP-puro reporter via CRISPR/Cas9-mediated homology-directed repair is generated via CRISPR/Cas9-mediated homology-directed repair.
This protocol covers both single-gRNA (sgRNA) and dual-gRNA directed knockout systems and describes the procedure for constructing an Auxin-Inducible Degron (AID) knock-in-mediated system to induce the rapid degradation of a target protein in cells, thereby investigating its function.
Qian Tang, Jian Liu· Methods in molecular biology· 0 citations
Human induced pluripotent stem cells (hiPSCs) represent a powerful platform for disease modeling, especially in monogenic diseases as they preserve the donor’s genetic background while enabling directed differentiation into disease-relevant cell types. This makes them highly suitable for studying disease mechanisms in a patient-specific and physiologically relevant context. Although CRISPR/Cas9 is widely applied for genome editing, precise correction of pathogenic variants in hiPSCs remains challenging due to the lack of standardized CRISPR component selection and experimental design. Here, we describe an optimized CRISPR-based strategy for correcting a heterozygous HNF1A frameshift mutation (c.235_236insG; p.Glu79Glyfs*16) in HNF1A-MODY patient-derived hiPSCs. Using electroporation, we efficiently delivered CRISPR components, including a ribonucleoprotein complex of Cas9 and single-guide RNA, along with a single-stranded oligodeoxynucleotide repair template. Corrected hiPSC lines were validated for pluripotency, absence of exogenous reprogramming factors, and off-target effects. Additionally, we discuss key technical challenges encountered during the editing process and provide practical recommendations that may improve the generation of mutation-corrected hiPSC lines. These guidelines could serve as a useful reference for researchers employing CRISPR-based strategies for generation of reliable disease modelling tools.
D. Skoczek, Jerzy Hohendorff, Maciej T. Małecki et al.· Human Genetics· 0 citations
The phenotypic profile of a CRISPR-generated Ent2 mutant line is described and the feasibility of combining genome editing with balancer chromosome strategies in Drosophila is demonstrated, demonstrating the feasibility of combining genome editing with balancer chromosome strategies in Drosophila.
Chikun Li, Lin Li, Ying Chen et al.· Journal of Visualized Experi...· 0 citations
It is reported that the choice of sgRNA target site affects its targeted cleavage activity and show that the combination of guideRNA with SpCas9 can effectively delete the exonic fragment of the SLC25A38 gene in HEK293T cells.
S. Yazdanparast, Hamid Galehdari, S. Khatami et al.· Journal of Applied Genetics· 0 citations
