CRISPR-based genome editing to endogenously distinguish the paralogs MAGOH and MAGOHB

IF 1 Q4 GENETICS & HEREDITY Gene Reports Pub Date : 2025-01-31 DOI:10.1016/j.genrep.2025.102152

Ayushi Rehman, Ajay Narwade, Kusum Kumari Singh

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Abstract

Paralogous genes have a high degree of similarity, making it challenging to discern their individual expression and function. Here, we describe a CRISPR-based method to distinguish highly similar paralog pairs endogenously. We employed this method to distinguish MAGOH and MAGOHB, a paralog pair found in the exon junction complex. MAGOH and MAGOHB differ by only two amino acids and cannot be distinguished at the protein level. However, synonymous substitutions in the coding sequence allow their distinction solely at the mRNA level. Using CRISPR-Cas9, we endogenously tagged MAGOH with the Myc epitope, enabling us to distinguish the two proteins in the HCT116 cell line. This tagging has allowed us to compare the stability of the paralogs at the mRNA and protein levels, providing new insights into the stability differences between the respective mRNA and protein. We also used this system to validate that the paralogs can simultaneously interact with other exon-junction components. The tagged cells offer a tool to study paralog-specific regulation, and the method can be employed to study other highly similar paralog pairs.

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来源期刊

Gene Reports Biochemistry, Genetics and Molecular Biology-Genetics

CiteScore

3.30

自引率

7.70%

发文量

246

审稿时长

49 days

期刊介绍： Gene Reports publishes papers that focus on the regulation, expression, function and evolution of genes in all biological contexts, including all prokaryotic and eukaryotic organisms, as well as viruses. Gene Reports strives to be a very diverse journal and topics in all fields will be considered for publication. Although not limited to the following, some general topics include: DNA Organization, Replication & Evolution -Focus on genomic DNA (chromosomal organization, comparative genomics, DNA replication, DNA repair, mobile DNA, mitochondrial DNA, chloroplast DNA). Expression & Function - Focus on functional RNAs (microRNAs, tRNAs, rRNAs, mRNA splicing, alternative polyadenylation) Regulation - Focus on processes that mediate gene-read out (epigenetics, chromatin, histone code, transcription, translation, protein degradation). Cell Signaling - Focus on mechanisms that control information flow into the nucleus to control gene expression (kinase and phosphatase pathways controlled by extra-cellular ligands, Wnt, Notch, TGFbeta/BMPs, FGFs, IGFs etc.) Profiling of gene expression and genetic variation - Focus on high throughput approaches (e.g., DeepSeq, ChIP-Seq, Affymetrix microarrays, proteomics) that define gene regulatory circuitry, molecular pathways and protein/protein networks. Genetics - Focus on development in model organisms (e.g., mouse, frog, fruit fly, worm), human genetic variation, population genetics, as well as agricultural and veterinary genetics. Molecular Pathology & Regenerative Medicine - Focus on the deregulation of molecular processes in human diseases and mechanisms supporting regeneration of tissues through pluripotent or multipotent stem cells.