TY - JOUR
T1 - Keratinocytes sense and eliminate CRISPR DNA through STING/IFN-κ activation and APOBEC3G induction
AU - Sarkar, Mrinal K.
AU - Uppala, Ranjitha
AU - Zeng, Chang
AU - Billi, Allison C.
AU - Tsoi, Lam C.
AU - Kidder, Austin
AU - Xing, Xianying
AU - White, Bethany E.Perez
AU - Shao, Shuai
AU - Plazyo, Olesya
AU - Sirobhushanam, Sirisha
AU - Xing, Enze
AU - Jiang, Yanyun
AU - Gallagher, Katherine A.
AU - Voorhees, John J.
AU - Michelle Kahlenberg, J.
AU - Gudjonsson, Johann E.
N1 - Funding Information:
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).
Publisher Copyright:
© 2023 American Society for Clinical Investigation. All rights reserved.
PY - 2023/5/1
Y1 - 2023/5/1
N2 - 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.
AB - 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.
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U2 - 10.1172/JCI159393
DO - 10.1172/JCI159393
M3 - Article
C2 - 36928117
AN - SCOPUS:85158818312
SN - 0021-9738
VL - 133
JO - Journal of Clinical Investigation
JF - Journal of Clinical Investigation
IS - 9
M1 - e159393
ER -