Keratinocytes sense and eliminate CRISPR DNA through STING/IFN-κ activation and APOBEC3G induction

Mrinal K. Sarkar, Ranjitha Uppala, Chang Zeng, Allison C. Billi, Lam C. Tsoi, Austin Kidder, Xianying Xing, Bethany E.Perez White, Shuai Shao, Olesya Plazyo, Sirisha Sirobhushanam, Enze Xing, Yanyun Jiang, Katherine A. Gallagher, John J. Voorhees, J. Michelle Kahlenberg, Johann E. Gudjonsson*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

CRISPR/Cas9 has been proposed as a treatment for genetically inherited skin disorders. Here we report that CRISPR transfection activates STING-dependent antiviral responses in keratinocytes, resulting in heightened endogenous interferon (IFN) responses through induction of IFN-κ, leading to decreased plasmid stability secondary to induction of the cytidine deaminase gene APOBEC3G. Notably, CRISPR-generated KO keratinocytes had permanent suppression of IFN-κ and IFNstimulated gene (ISG) expression, secondary to hypermethylation of the IFNK promoter region by the DNA methyltransferase DNMT3B. JAK inhibition via baricitinib prior to CRISPR transfection increased transfection efficiency, prevented IFNK promoter hypermethylation, and restored normal IFN-κ activity and ISG responses. This work shows that CRISPR-mediated gene correction alters antiviral responses in keratinocytes, has implications for future gene therapies for inherited skin diseases using CRISPR technology, and suggests pharmacologic JAK inhibition as a tool for facilitating and attenuating inadvertent selection effects in CRISPR/Cas9 therapeutic approaches.

Original languageEnglish (US)
Article numbere159393
JournalJournal of Clinical Investigation
Volume133
Issue number9
DOIs
StatePublished - May 1 2023

Funding

We thank Kathleen J. Green and Lisa M. Godsel (Department of Dermatology, Northwestern University, Chicago, Illinois, USA) for providing the primary human keratinocytes to study the transfection efficiency. This work was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the NIH under award numbers R01-AR060802 (to JEG), P30-AR075043 (to JEG, LCT, MKS, and JMK), R01-AR069071 (to JEG), R01-AR071384 (to JMK), K24-AR076975 (to JMK), R21-AR077741 (to JEG), and K01-AR072129 (to LCT), National Psoriasis Foundation Translational Research Grant 852098 (to MKS), the A. Alfred Taubman Medical Research Institute (to JEG and JMK), and the Parfet Emerging Scholar Award (to JMK). This work was also supported by the National Psoriasis Foundation Psoriasis Prevention Initiative and the Dermatology Foundation (to JEG).

ASJC Scopus subject areas

  • General Medicine

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