Abstract
The regulation of protein stability is a fundamental issue for biophysical processes, but there has not previously been a convenient and unbiased method of identifying regulators of protein stability. However, as reported in the article entitled “A genome-scale CRISPR–Cas9 screening method for protein stability reveals novel regulators of Cdc25A,” recently published in Cell Discovery, our team developed a protein stability regulators screening assay (Pro-SRSA) by combining the whole-genome clustered regularly interspaced short palindromic repeats Cas9 (CRISPR–Cas9) library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability. Based on our findings, we are confident that this efficient and unbiased screening method at the genome scale will be used by researchers worldwide to identify regulators of protein stability.
Highlights
Fine-tuned degradation of proteins plays a crucial role in a variety of physiological conditions, including cell cycle progression, cell growth, differentiation, and signaling transduction
Based on the study reported in the article entitled “A genome-scale CRISPR–Cas9 screening method for protein stability reveals novel regulators of cell division cycle 25A (Cdc25A),” which was published in Cell Discovery, such an efficient method has been developed [2]
By combining the CRISPR–Cas9 library and the dual-fluorescence reporter, we were able to sort the cells with top ratio of enhanced green fluorescence protein (EGFP)/ DsRed, extract the genome DNA of the sorted and unsorted control cells and, by polymerase chain reaction, amplify the single-guide RNAs (sgRNAs)
Summary
Fine-tuned degradation of proteins plays a crucial role in a variety of physiological conditions, including cell cycle progression, cell growth, differentiation, and signaling transduction. Understanding the precise regulation of protein degradation, especially for oncogenes and tumor suppressors, is important for understanding the biological mechanisms of cancer and for developing therapeutic interventions. Due to the lack of an efficient method, an unbiased and universal delineation of the regulators of protein homeostasis has not been previously possible.
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