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

IF 0.9 Q4 GENETICS & HEREDITY Gene Reports Pub Date : 2025-03-01 Epub 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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基于crispr的基因组编辑，内源性区分MAGOH和MAGOHB的相似物

同源基因具有高度的相似性，这使得辨别它们的个体表达和功能具有挑战性。在这里，我们描述了一种基于crispr的方法来内源性区分高度相似的平行对。我们使用这种方法来区分MAGOH和MAGOHB，这是一个在外显子连接复合体中发现的平行对。MAGOH和MAGOHB只有两个氨基酸不同，在蛋白质水平上不能区分。然而，编码序列中的同义替换允许它们仅在mRNA水平上进行区分。使用CRISPR-Cas9，我们用Myc表位内源性标记MAGOH，使我们能够在HCT116细胞系中区分这两种蛋白。这种标记使我们能够比较类似物在mRNA和蛋白质水平上的稳定性，为各自mRNA和蛋白质之间的稳定性差异提供新的见解。我们还使用该系统验证了类似物可以同时与其他外显子结组分相互作用。标记的细胞提供了一种研究平行特异性调控的工具，该方法可用于研究其他高度相似的平行对。

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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.