We present here a protocol for biallelic tagging of an endogenous gene in human cells using CRISPR-Cas9 editing technology. Using RIF1 as an example, we describe tagging the gene with a mini-auxin-inducible degron and a green fluorescent protein at its C terminus. We detail steps for preparing and designing the sgRNA and homologous repair template, and clone selection and verification. For complete details on the use and execution of this protocol, please refer to Kong et al..
Download full-text PDF |
Source |
|---|---|
| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC10241970 | PMC |
| http://dx.doi.org/10.1016/j.xpro.2023.102286 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
PATL2 mutations affect human oocyte maternal mRNA homeostasis and protein interactions in cell cycle regulation.
Cell Biosci
December 2024
Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, 310016, China.
Background: Oocyte maturation defect (OMD) and early embryonic arrest result in female infertility. Previous studies have linked biallelic mutations in the PATL2 gene to OMD, yet the underlying mechanism remains largely unknown.
Results: This study uncovers three novel mutations (c.
CRISPR/Cas9 has massively accelerated the generation of gene loss-of-function models in zebrafish. However, establishing tissue-specific mutant lines remains a laborious and time-consuming process. Although a few dozen tissue-specific Cas9 zebrafish lines have been developed, the lack of standardization of some key methods, including gRNA delivery, has limited the implementation of these approaches in the zebrafish community.
View Article and Find Full Text PDFA therapeutic approach to pantothenate kinase associated neurodegeneration: a pilot study.
Orphanet J Rare Dis
November 2024
Andalusian Centre for Developmental Biology-CSIC-Pablo de Olavide University, 41013, Seville, Spain.
Article Synopsis
- * The diagnosis of PKAN relies on clinical observations, a specific brain MRI finding called the "eye of the tiger," and genetic testing for mutations in the pantothenate kinase 2 (PANK2) gene, which plays a crucial role in coenzyme A (CoA) production.
- * Research shows that combining multitarget supplements (like pantothenate, pantethine, omega-3, and vitamin E) with standard
Introducing a novel TRAPPC10 gene variant as a potential cause of developmental delay and intellectual disability in an Iranian family.
Neurogenetics
November 2024
Department of Medical Genetics, School of Medicine, Ilam University of Medical sciences, Ilam, Iran.
Background: TRAPP complexes are crucial components for intracellular transport and cellular organization. Their role in vesicle trafficking, particularly through their involvement in the secretory pathway, make them more important in neurodevelopmental mechanisms. This study aims to identify a novel genetic variant, associated with developmental delay and intellectual disability by analyzing a consanguineous Iranian family.
View Article and Find Full Text PDFDevelopment of an optimized protocol for generating knockout cancer cell lines using the CRISPR/Cas9 system, with emphasis on transient transfection.
PLoS One
November 2024
Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Microbiology Research Center, Tehran, Iran.
The clustered regularly interspaced short palindromic repeats (CRISPR) system offers cost-effectiveness, high efficiency, precision, and ease of use compared to traditional gene editing techniques. In this study, we employed findings from prestigious investigations to develop an optimized approach for generating knockout cancer cell lines using a transient transfection method. This protocol introduces a distinctive approach that follows rigorous guidelines for designing gRNA to reduce off-target effects, a major challenge in CRISPR applications.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!