Work

Comprehensive CRISPR/Cas9-based mutagenesis identifies single-amino acid substitutions that abrogate SPEN function in X inactivation

Year: 2026

Type: article

Abstract: While genetic screens have facilitated the dissection of protein function in animal development, advances in systematic point mutagenesis open new opportunities for forward genetics in mammalian cells. Here, we develop a CRISPR/Cas9-mediated base editing screen that allows functional screening of extensive collections of single amino acid substitutions of endogenous proteins. We demonstrate the application on the X-chromosomal Hprt and the autosomal Msh2 gene in diploid male and haploid mouse embryonic stem cells, respectively. Finally, we use this methodology to generate a sequence-function map of the transcriptional co-repressor SPEN in X chromosome inactivation. We demonstrate that the substitution of the SPEN RRM4-residue W522 abrogates X-linked gene repression by Xist RNA and impairs the establishment of H3K27me3 deposition. Our results demonstrate that screening in haploid cells allows efficient identification of mutations that would be recessive in diploid cells, suggesting applications across a wide range of areas.

Source: Nature Communications

DOI: 10.1038/s41467-026-71400-4

Authors: Corinne Kaufmann, Sarah Sting, Chao Dai, Anton Wutz

Institutions: ETH Zurich, Institute of Molecular Health Sciences

Language: English

Topic: CRISPR and Genetic Engineering

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $6,990

Funder: Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung