title: A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A.
abstract: The regulation of stability is particularly crucial for unstable proteins in cells. However, a convenient and unbiased method of identifying regulators of protein stability remains to be developed. Recently, a genome-scale CRISPR-Cas9 library has been established as a genetic tool to mediate loss-of-function screening. Here, we developed a protein stability regulators screening assay (Pro-SRSA) by combining the whole-genome CRISPR-Cas9 library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability. Using Cdc25A as an example, Cul4B-DDB1(DCAF8) was identified as a new E3 ligase for Cdc25A. Moreover, the acetylation of Cdc25A at lysine 150, which was acetylated by p300/CBP and deacetylated by HDAC3, prevented the ubiquitin-mediated degradation of Cdc25A by the proteasome. This is the first study to report that acetylation, as a novel posttranslational modification, modulates Cdc25A stability, and we suggest that this unbiased CRISPR-Cas9 screening method at the genome scale may be widely used to globally identify regulators of protein stability.
authors.affiliation: Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine , Guangzhou, China.
authors.forename: Yuanzhong
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine , Guangzhou, China.
authors.forename: Liwen
authors.initials: L
authors.lastname: Zhou
authors.affiliation: Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine , Guangzhou, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Wang
authors.affiliation: Clinical Laboratory and Medical Research Center, Zhuhai Hospital, Jinan University, Zhuhai People's Hospital , Zhuhai, China.
authors.forename: Jinping
authors.initials: J
authors.lastname: Lu
authors.affiliation: Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine , Guangzhou, China.
authors.forename: Ruhua
authors.initials: R
authors.lastname: Zhang
authors.affiliation: Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine , Guangzhou, China.
authors.forename: Xiaoting
authors.initials: X
authors.lastname: Liang
authors.affiliation: Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine , Guangzhou, China.
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.affiliation: Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine , Guangzhou, China.
authors.forename: Wuguo
authors.initials: W
authors.lastname: Deng
authors.affiliation: Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine , Guangzhou, China.
authors.forename: Yi-Xin
authors.initials: YX
authors.lastname: Zeng
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine , Rochester, MN, USA.
authors.forename: Haojie
authors.initials: H
authors.lastname: Huang
authors.affiliation: Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou, China; Guangdong Provincial Key Laboratory of Tumor Targeted Drugs, Guangzhou Doublle Bioproducts Co. Ltd., Guangzhou, China.
authors.forename: Tiebang
authors.initials: T
authors.lastname: Kang
keywords: CRISPR-Cas9 screening; Cdc25A; acetylation; protein stability; ubiquitination