Biallelic and gene-wide genomic substitution for endogenous intron and retroelement mutagenesis in human cells

Tomoyuki Ohno, Taichi Akase, Shunya Kono, Hikaru Kurasawa, Takuto Takashima, Shinya Kaneko and Yasunori Aizawa ()
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Tomoyuki Ohno: Tokyo Institute of Technology
Taichi Akase: Tokyo Institute of Technology
Shunya Kono: Tokyo Institute of Technology
Hikaru Kurasawa: Tokyo Institute of Technology
Takuto Takashima: Tokyo Institute of Technology
Shinya Kaneko: Tokyo Institute of Technology
Yasunori Aizawa: Tokyo Institute of Technology

Nature Communications, 2022, vol. 13, issue 1, 1-13

Abstract: 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.

Date: 2022
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DOI: 10.1038/s41467-022-31982-1

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