RRC ID 82052
Author Ohno T, Akase T, Kono S, Kurasawa H, Takashima T, Kaneko S, Aizawa Y.
Title Biallelic and gene-wide genomic substitution for endogenous intron and retroelement mutagenesis in human cells.
Journal Nat Commun
Abstract Functional annotation of the vast noncoding landscape of the diploid human genome still remains a major challenge of genomic research. An efficient, scarless, biallelic, and gene-wide mutagenesis approach is needed for direct investigation of the functional significance of endogenous long introns in gene regulation. Here we establish a genome substitution platform, the Universal Knock-in System or UKiS, that meets these requirements. For proof of concept, we first used UKiS on the longest intron of TP53 in the pseudo-diploid cell line HCT116. Complete deletion of the intron, its substitution with mouse and zebrafish syntenic introns, and specific removal of retrotransposon-derived elements (retroelements) were all efficiently and accurately achieved in both alleles, revealing a suppressive role of intronic Alu elements in TP53 expression. We also used UKiS for TP53 intron deletion in human induced pluripotent stem cells without losing their stemness. Furthermore, UKiS enabled biallelic removal of all introns from three human gene loci of ~100 kb and longer to demonstrate that intron requirements for transcriptional activities vary among genes. UKiS is a standard platform with which to pursue the design of noncoding regions for genome writing in human cells.
Volume 13(1)
Pages 4219
Published 2022-7-21
DOI 10.1038/s41467-022-31982-1
PII 10.1038/s41467-022-31982-1
PMID 35864085
PMC PMC9304424
MeSH Animals Genomics Humans Induced Pluripotent Stem Cells* Introns / genetics Mice Mutagenesis / genetics Retroelements* / genetics Zebrafish / genetics
Resource
Human and Animal Cells 201B7(HPS0063)