2023 ACMG Annual Clinical Genetics Meeting Digital Edition: Poster Gallery-Efficient allelic tagging in human induced pluripotent stem cells using CRISPR

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This document describes a method for efficiently tagging protein-coding genes with fluorescent reporters in human induced pluripotent stem cells (iPSCs) using CRISPR/Cas9-mediated homology-directed repair. The protocol outlines steps for designing the single guide RNA (sgRNA) and donor vector, preparing and maintaining the iPSCs, synthesizing the sgRNA, delivering the Cas9/sgRNA complex and donor vectors into the cells using nucleofection, verifying the allelically tagged clones using genotyping PCR, and validating the expression of fluorescent reporters and pluripotency markers using flow cytometry.<br /><br />The protocol yields iPSC clones with bi-allelically tagged clones of the gene-of-interest, with a 0.1% positive rate of human iPSCs having both alleles differentially tagged. Single cells are sorted into individual wells to generate clones, resulting in an average of 30 clones per plate.<br /><br />The method allows for real-time gene expression monitoring and protein subcellular localization in an allele-specific manner, characterization of cis vs. trans regulatory element effects on gene expression, and facilitation of pooled CRISPR screening to identify essential genetic components for target gene expression and associated phenotypes.<br /><br />It should be noted that this strategy is not suitable for silent genes, but the addition of a promoter combined with the Cre-Lox system may address this limitation.<br /><br />In conclusion, the described method provides an efficient approach for bi-allelic tagging of protein-coding genes with fluorescent reporters in human iPSCs, enabling allele-specific investigation of transcriptional regulation and identification of genetic components important for gene expression and phenotypes.

Asset Subtitle

Co-Author - Xingjie Ren, PhD; Presenting Author - Maya Takagi, BA; Co-Author - Yin Shen, PhD;

Meta Tag

Biochemical genetics

Genomic Methodologies

Co-Author Xingjie Ren, PhD

Co-Author Yin Shen, PhD

Presenting Author Maya Takagi, BA

Keywords

protein-coding genes

fluorescent reporters

human induced pluripotent stem cells

CRISPR/Cas9-mediated homology-directed repair

single guide RNA

donor vector

nucleofection

genotyping PCR

flow cytometry

bi-allelic tagging